Tree Physiology

Ülo Niinemets
Russell K. Monson Editors

Biology, Controls
and Models of Tree
Volatile Organic
Compound
Emissions
Biology, Controls and Models of Tree Volatile
Organic Compound Emissions
Tree Physiology
Volume 5

Series Editors: Frederick C. Meinzer and Ülo Niinemets

For further volumes:

http://www.springer.com/series/6644
Ülo Niinemets • Russell K. Monson
Editors

Biology, Controls and

Models of Tree Volatile
Organic Compound
Emissions

123
Editors
Ülo Niinemets Russell K. Monson
Institute of Agricultural and Environmental School of Natural Resources and the
Sciences Environment
Estonian University of Life Sciences University of Arizona
Tartu, Estonia Tucson, AZ, USA

ISSN 1568-2544
ISBN 978-94-007-6605-1 ISBN 978-94-007-6606-8 (eBook)
DOI 10.1007/978-94-007-6606-8
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Preface

Ülo Niinemets and Russell K. Monson

Discoveries that plants are in part responsible for the blue haze commonly observed
in the atmosphere above many forests (Went 1960; Rasmussen and Went 1965) and
for the tropospheric ozone pollution in many forested urban and suburban areas
(Rasmussen 1972; Chameides et al. 1988) have compelled researchers to ask what
are the plants emitting, how much is being emitted and how do these emissions
impact our environment? These very important questions are at the heart of a highly
interdisciplinary research field: biogenic volatile organic compounds (BVOC) in the
biosphere–atmosphere component of the Earth system.
Fritz Went, a plant physiologist famous for discoveries on plant growth regu-
lators, was also intrigued by the potential for plants to form atmospheric hazes
above forests. He hypothesized that the organic compounds emitted from plants,
many of which he could detect with his own sense of smell, contributed to this
haze. In 1960, he made measurements on the mass of leaf oils in shrubs from the
Western United States and estimated global terpene emissions to be 175 Tg C year1
(1 Tg D 1012 g) (Went 1960). Since that seminal study, estimates of global BVOC
emissions (excluding methane) have been refined through coupled vegetation–
atmosphere models and better maps of global vegetation. Currently, the worldwide
emissions are estimated to be around 1 Pg C year1 (1 Pg D 1015 g) (Guenther
et al. 2012). Much of the past research on plant volatile emissions has been, and
continues to be, on the simple C5 volatile hydrocarbon, isoprene, which is emitted
from leaves in a light- and temperature-dependent manner and is coupled to the
metabolic processes of photosynthesis (Sanadze 1956; Rasmussen and Went 1965;
Rasmussen 1970). Since its initial discovery, the research on biogenic isoprene has

Ü. Niinemets
Department of Plant Physiology, Institute of Agricultural and Environmental Sciences,
Estonian University of Life Sciences, Tartu, Estonia
R.K. Monson
School of Natural Resources and the Environment and the Laboratory for Tree Ring Research,
University of Arizona, Tucson, AZ 85721, USA

v
vi Preface

focused on understanding its source strength and distribution among different plant
taxa, resulting in the construction of first list of isoprene-emitting plants (Rasmussen
1978) and first biogenic emission inventory system (BEIS, Pierce and Waldruff
1991), followed by more biologically oriented emission algorithms and inventories
(e.g., Guenther et al. 1991, 1994, 2006, 2012; Arneth et al. 2007; Monson et al.
2012). By now, it is widely acknowledged that multiple plant BVOC emissions play
a major role in atmospheric dynamics within the Earth system with highly reactive
compounds reacting in the gas phase to affect atmospheric chemistry, including
influences on less reactive compounds that affect the atmosphere’s global warming
potential, e.g., methane (Fuentes et al. 2000). Furthermore, condensation of photo-
oxidation products of BVOCs leads to the formation of secondary organic aerosols,
which also generate cloud condensation nuclei and have profound implications for
the Earth’s solar radiation budget and climate (Kulmala et al. 2004; Huff Hartz et
al. 2005; Hallquist et al. 2009; Arneth et al. 2010). Trees have been traditionally
thought to contribute the most to BVOC emissions due to the presence of multiple
tree species with high emission rates (e.g., the oaks and poplars) and large aerial
coverage, which gives forests a unique global role in regulating atmospheric
chemistry. Apart from strong constitutive emitters, emissions induced by abiotic
and biotic stresses have also been identified in many important forest species
previously considered “non-emitters” (Holopainen and Gershenzon 2010; Loreto
and Schnitzler 2010; Niinemets 2010), implying that there are strong biological
controls on the emissions of BVOCs that we still do not understand and that continue
to cause large uncertainties in our models and inventories.
From a biological perspective, the key question has been why do plants emit
these volatiles? What are the costs and benefits for the plant, and why do some
plant species emit these compounds constitutively and in others the emissions must
be induced? The role of isoprene emissions was unknown until the mid-1990s
when it was discovered that isoprene enhances plant thermotolerance (Sharkey and
Singsaas 1995) and increases the resistance of plant metabolism to atmospheric
oxidants (Loreto et al. 2001), followed by the development of a hypothesis of
general enhancement of cellular oxidative stress tolerance (Vickers et al. 2009a).
Lately, breakthroughs in the genetic engineering of plants have allowed us to test
these hypotheses with unprecedented specificity in the processes affected (Behnke
et al. 2007, 2010; Vickers et al. 2009b; Velikova et al. 2011). Rapid developments
in the availability of genomic information have also recently allowed researchers to
gain insight into evolutionary patterns and past influences by natural selection on the
isoprene emission capacity (Monson et al. 2013; Sharkey et al. 2013). Furthermore,
the role of multiple plant BVOCs in abiotic and biotic stress signalling within and
among plants, and among plants and insects has drawn strong attention from a
broader complement of the ecological research community (Owen and Peñuelas
2005; Dicke et al. 2009; Dicke and Baldwin 2010; Holopainen and Gershenzon
2010).
In the past, three comprehensive treatises have been published on trace gas
emission from vegetation (Sharkey et al. 1991; Hewitt 1999; Gasche et al. 2003).
Only the book by Sharkey et al. (1991) deals exclusively with biological controls
Preface vii

on BVOC, while the book by Hewitt (1999) focuses on quantification of emissions,

and the book by Gasche et al. (2003) only briefly considers biological controls.
Since the publication of these books, there has been rapid progress in understanding
the controls over isoprene emissions, including the discovery of isoprene synthase
(Silver and Fall 1991), its sequence in several organisms (e.g., Miller et al.
2001; Vickers et al. 2010; Sharkey et al. 2013) and establishment of the reaction
mechanism (Köksal et al. 2010). Furthermore, there has been a significant increase
in our knowledge of multiple terpene synthase structures and regulation (Bohlmann
et al. 1998; Fischbach et al. 2001; Degenhardt et al. 2009; Chen et al. 2011;
Keeling et al. 2011; Köksal et al. 2011). Due to the development of sensors and
detection technology that was in its infancy at the time of the appearance of the
book edited by Hewitt (1999), a large volume of literature has emerged on BVOC
flux quantifications (Karl et al. 2002, 2007; Müller et al. 2010). Finally, since the
publication of the three past books, the field of BVOC emissions has matured greatly
in our understanding of BVOC emission models and their connection to underlying
metabolic processes (Monson et al. 2012).
This book intends to cover all biological scales of organization from molecular
to globe and to make a major leap in summarizing and synthesizing the existing
information accumulated since the publication of the past comprehensive summaries
of BVOC emissions. The book consists of 17 primary chapters followed by a
synthesis. The chapters focus on four major topics: evolutionary diversification
and perspectives for genetic engineering of volatile organic compound emissions
(chapters 1–4), controls over emissions (chapters 5–7), emissions under stress
(chapters 8–11), and emission models (chapters 12–17).
Chapters 1–4 cover tree BVOC emission diversity and evolutionary aspects as
driven by abiotic (chapter of Fineschi et al.) and biotic (chapter of Trowbridge
and Stoy) stresses, and molecular diversity (chapters of Rajabi Memari et al.
and Rosenkranz and Schnitzler), specifically asking how and why the capacity
for constitutive volatile production has evolved, what are the key biochemical
pathways and factors that determine the blend of emitted volatiles. Here also the
diversity in elicited emissions (chapter of Trowbridge and Stoy) and ways of genetic
modification to alter emissions (chapter of Rosenkranz and Schnitzler) are analysed.
Chapters 5–7 provide an overview of the cellular and leaf-level mechanisms
controlling BVOC emissions. The group of these chapters starts with short-term
molecular pathway and leaf-level metabolic controls (chapters of Li and Sharkey,
and Monson), then covers longer-term controls by carbon availability and gene
expression level acclimation responses to environmental variability (chapter of
Monson) and finishes with stomatal and physico-chemical controls driven by
variations in compound volatility (chapter of Harley).
Chapters 8–11 review the effects of abiotic and biotic stresses on volatile emis-
sions. Consideration of this topic starts with a chapter analysing the modification
of tree abiotic stress tolerance by the capacity for volatile emissions, emphasizing
tolerance to heat, drought, salinity and resulting oxidative stress (chapter of Possell
and Loreto). Then responses of emissions to flooding (chapter of Kreutzwieser and
Rennenberg) and air pollution and elevated [CO2 ] (chapter of Calfapietra et al.)
viii Preface

are reviewed. This topic is concluded with a chapter investigating the multitrophic
interactions between trees, herbivores and herbivore enemies in future polluted
atmospheres that significantly alter the role of volatile compounds as important
ecological signals that organize food webs and host parasite relations (chapter of
Holopainen et al.).
The chapters 12–17 put the information summarized in previous chapters into
the quantitative framework of predictive emission models. This topic starts with a
review of leaf-level emission algorithms (chapter of Grote et al.), then covers canopy
and landscape (chapters of Niinemets et al. and Guenther) and biome level models
(chapter of Ashworth et al.), emphasizing the philosophy of model parameterization
and validation. Finally, global feedbacks to climate and atmospheric composition
due to tree BVOC emissions are analysed (chapter of Kulmala et al.).
The book concludes with a synthesis section that puts the contents of the book
into global biosphere–atmosphere science context and provides a perspective for
future developments in the field. In addition to summarizing the state-of-the-art
information of tree volatile emissions, the book, in particular, explores the ways
that rich archives of molecular, physiological and ecological evidence can be best
included in quantitative emission models. The content and focus of the chapters
are intended for use by graduate students, researchers and policy professionals
interested in the recent developments in the field. As in many interdisciplinary,
complex and rich science fields, the challenge in BVOC research is to get to
an intellectual place beyond the fragmented, and increasingly more detailed,
knowledge provided in the primary literature to one of synthetic understanding,
utility for framing further hypotheses and application to devising observation and
regulation policies that improve the global human condition. We hope that this book
can serve as a springboard for those interested in starting a career in BVOC research,
those continuing to pursue research at the frontier of this fascinating field and those
interested in applying the research to the better management of our Earth system.

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Contents

1 Diversification of Volatile Isoprenoid Emissions from

Trees: Evolutionary and Ecological Perspectives . . .. . . . . . . . . . . . . . . . . . . . 1
Silvia Fineschi, Francesco Loreto, Michael Staudt,
and Josep Peñuelas
2 BVOC-Mediated Plant-Herbivore Interactions . . . . .. . . . . . . . . . . . . . . . . . . . 21
Amy M. Trowbridge and Paul C. Stoy
3 The Biochemistry and Molecular Biology of Volatile
Messengers in Trees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 47
Hamid Rajabi Memari, Leila Pazouki, and Ülo Niinemets
4 Genetic Engineering of BVOC Emissions from Trees . . . . . . . . . . . . . . . . . . 95
Maaria Rosenkranz and Jörg-Peter Schnitzler
5 Molecular and Pathway Controls on Biogenic Volatile
Organic Compound Emissions . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 119
Ziru Li and Thomas D. Sharkey
6 Metabolic and Gene Expression Controls
on the Production of Biogenic Volatile Organic Compounds . . . . . . . . . . 153
Russell K. Monson
7 The Roles of Stomatal Conductance and Compound
Volatility in Controlling the Emission of Volatile Organic
Compounds from Leaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 181
Peter C. Harley
8 The Role of Volatile Organic Compounds in Plant
Resistance to Abiotic Stresses: Responses and Mechanisms . . . . . . . . . . . 209
Malcolm Possell and Francesco Loreto
9 Flooding-Driven Emissions from Trees . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 237
Jürgen Kreuzwieser and Heinz Rennenberg

xi
xii Contents

10 Modification of BVOC Emissions by Changes

in Atmospheric [CO2 ] and Air Pollution . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 253
Carlo Calfapietra, Emanuele Pallozzi, Ilaria Lusini,
and Violeta Velikova
11 Multitrophic Signalling in Polluted Atmospheres. . .. . . . . . . . . . . . . . . . . . . . 285
Jarmo K. Holopainen, Anne-Marja Nerg,
and James D. Blande
12 Leaf-Level Models of Constitutive and Stress-Driven
Volatile Organic Compound Emissions . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 315
Rüdiger Grote, Russell K. Monson, and Ülo Niinemets
13 Scaling BVOC Emissions from Leaf to Canopy
and Landscape: How Different Are Predictions Based
on Contrasting Emission Algorithms? . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 357
Ülo Niinemets, Paolo Ciccioli, Steffen M. Noe,
and Markus Reichstein
14 Upscaling Biogenic Volatile Compound Emissions
from Leaves to Landscapes . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 391
Alex Guenther
15 Scaling Emissions from Agroforestry Plantations
and Urban Habitats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 415
Susan M. Owen, C. Nicholas Hewitt, and Clare S. Rowland
16 Global Modelling of Volatile Organic Compound Emissions . . . . . . . . . . 451
Kirsti Ashworth, Christophe Boissard, Gerd Folberth,
Juliette Lathière, and Guy Schurgers
17 Climate Feedbacks Linking the Increasing Atmospheric
CO2 Concentration, BVOC Emissions, Aerosols
and Clouds in Forest Ecosystems . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 489
Markku Kulmala, Tuomo Nieminen, Robert Chellapermal,
Risto Makkonen, Jaana Bäck, and Veli-Matti Kerminen
18 State-of-the-Art of BVOC Research: What Do We Have
and What Have We Missed? A Synthesis . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 509
Ülo Niinemets and Russell K. Monson

Editors Biography .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 529

Index . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 531
Contributors

Kirsti Ashworth Institute for Meteorology and Climate Research – Atmospheric

Environmental Research (IMK-IFU), Karlsruhe Institute for Technology (KIT),
Garmisch-Partenkirchen, Germany
Jaana Bäck Department of Physics, University of Helsinki, Helsinki, Finland
Department of Forest Sciences, University of Helsinki, Helsinki, Finland
James D. Blande Department of Environmental Science, University of Eastern
Finland, Kuopio, Finland
Christophe Boissard Laboratoire des Sciences du Climat et de l’Environnement –
LSCE-IPSL, CEA-CNRS-UVSQ, Gif-sur-Yvette, France
Carlo Calfapietra Institute of Agro-Environmental and Forest Biology (IBAF),
Consiglio Nazionale delle Ricerche (CNR), Porano, TR, Italy
Robert Chellapermal Department of Physics, University of Helsinki, Helsinki,
Finland
Paolo Ciccioli Istituto di Metodologie Chimiche, Consiglio Nazionale delle
Ricerche (CNR), Monterotondo Scalo, Italy
Silvia Fineschi Istituto per la Protezione delle Piante (IPP), Consiglio Nazionale
delle Ricerche (CNR), Sesto Fiorentino, FI, Italy
Gerd Folberth Met Office Hadley Centre, Exeter, Devon, UK
Rüdiger Grote Institute for Meteorology and Climate Research – Atmospheric
Environmental Research (IMK-IFU), Karlsruhe Institute for Technology (KIT),
Garmisch-Partenkirchen, Germany
Alex Guenther Biosphere-Atmosphere Interactions Group, Atmospheric
Chemistry Division, Earth System Laboratory, National Center for Atmospheric
Research, Boulder, CO, USA

xiii
xiv Contributors

Peter C. Harley Biosphere-Atmosphere Interactions Group, Atmospheric

Chemistry Division, Earth System Laboratory, National Center for Atmospheric
Research, Boulder, CO, USA
C. Nicholas Hewitt Lancaster Environment Centre, Lancaster University,
Lancaster, UK
Jarmo K. Holopainen Department of Environmental Science, University of
Eastern Finland, Kuopio, Finland
Veli-Matti Kerminen Department of Physics, University of Helsinki, Helsinki,
Finland
Jürgen Kreuzwieser Professur für Baumphysiologie, Albert-Ludwigs-Universität
Freiburg, Freiburg im Breisgau, Germany
Markku Kulmala Department of Physics, University of Helsinki, Helsinki,
Finland
Juliette Lathière Laboratoire des Sciences du Climat et de l’Environnement –
LSCE-IPSL, CEA-CNRS-UVSQ, Gif-sur-Yvette, France
Ziru Li Department of Biochemistry and Molecular Biology, Michigan State
University, East Lansing, MI, USA
Francesco Loreto Istituto per la Protezione delle Piante (IPP), Consiglio
Nazionale delle Ricerche (CNR), Sesto Fiorentino, FI, Italy
Ilaria Lusini Institute of Agro-Environmental and Forest Biology (IBAF),
Consiglio Nazionale delle Ricerche (CNR), Porano, TR, Italy
Department for Innovation in Biological, Agro-Food and Forest Systems, University
of Tuscia, Viterbo, Italy
Risto Makkonen Department of Physics, University of Helsinki, Helsinki, Finland
Hamid Rajabi Memari Genetic Engineering and Molecular Genetics, Biotechnol-
ogy and Life Science Center and School of Agriculture, Shahid Chamran University
of Ahvaz, Ahvaz, Iran
Russell K. Monson School of Natural Resources and the Environment and the
Laboratory for Tree Ring Research, University of Arizona, Tucson, AZ, USA
Anne-Marja Nerg Department of Environmental Science, University of Eastern
Finland, Kuopio, Finland
Tuomo Nieminen Department of Physics, University of Helsinki, Helsinki,
Finland
Ülo Niinemets Department of Plant Physiology, Institute of Agricultural and
Environmental Sciences, Estonian University of Life Sciences, Tartu, Estonia
Contributors xv

Steffen M. Noe Department of Plant Physiology, Institute of Agricultural and

Environmental Sciences, Estonian University of Life Sciences, Tartu, Estonia
Susan M. Owen Biosphere Atmosphere Interactions, Centre for Ecology and
Hydrology, Penicuik, Midlothian, UK
Emanuele Pallozzi Institute of Agro-Environmental and Forest Biology (IBAF),
Consiglio Nazionale delle Ricerche (CNR), Porano, TR, Italy
Leila Pazouki Institute of Agricultural and Environmental Sciences, Estonian
University of Life Sciences, Tartu, Estonia
Josep Peñuelas CSIC, Global Ecology Unit, CREAF-CEAB-UAB, Catalonia,
Spain
CREAF, Catalonia, Spain
Malcolm Possell Faculty of Agriculture and Environment, University of Sydney,
Sydney, NSW, Australia
Markus Reichstein Max Planck Institute for Biogeochemistry, Jena, Germany
Heinz Rennenberg Professur für Baumphysiologie, Albert-Ludwigs-Universität
Freiburg, Freiburg im Breisgau, Germany
Maaria Rosenkranz Research Unit Environmental Simulation (EUS), Institute of
Biochemical Plant Pathology, Helmholtz Zentrum München, Neuherberg, Germany
Clare S. Rowland Monitoring and Observation Systems, Centre for Ecology and
Hydrology, Lancaster, UK
Jörg-Peter Schnitzler Research Unit Environmental Simulation (EUS),
Institute of Biochemical Plant Pathology, Helmholtz Zentrum München,
Neuherberg, Germany
Guy Schurgers Department of Physical Geography and Ecosystem Science,
Lund University, Lund, Sweden
Thomas D. Sharkey Department of Biochemistry and Molecular Biology,
Michigan State University, East Lansing, MI, USA
Michael Staudt Centre d’Ecologie Fonctionnelle et Evolutive, UMR 5175,
Montpellier cedex 5, France
Paul C. Stoy Department of Land Resources and Environmental Sciences,
Montana State University, Bozeman, MT, USA
Amy M. Trowbridge Department of Land Resources and Environmental Sciences,
Montana State University, Bozeman, MT, USA
Violeta Velikova Institute of Plant Physiology and Genetics, Bulgarian Academy
of Sciences, Sofia, Bulgaria
Chapter 1
Diversification of Volatile Isoprenoid
Emissions from Trees: Evolutionary
and Ecological Perspectives

Silvia Fineschi, Francesco Loreto, Michael Staudt, and Josep Peñuelas

Abstract Not all tree species are strong constitutive volatile compound emitters,
and a variety of hypotheses have been put forward to explain the evolution and
the function of the emissions of volatile compounds. This chapter reviews the
evolutionary and ecological aspects of volatile compound production in trees,
specifically asking how and in which tree species the capacity for constitutive
volatile production has evolved. The capacity for volatile emissions is a polyphyletic
trait present in several diverse plant groups, but the presence of emission capacity
is not directly related to phylogenetic distance among the species and species
genera, demonstrating that the trait has evolved multiple times during evolution.
We here review present volatile emission inventories highlighting the need for more
worldwide, coordinated efforts to obtain realistic data of geographical and taxo-
nomic patterns. We thereafter discuss the past evolution of isoprenoid emissions,
and pose the questions of why isoprene emission is particularly widespread in
hygrophytes, why it is a characteristic of mostly fast-growing perennial plants and
why it is stimulated by low concentrations of CO2 . Finally, we discuss the future,
how climate and global change and the corresponding ecological constraints impact
the diversification and emission of volatile organic compounds from plants.

S. Fineschi () • F. Loreto

Istituto per la Protezione delle Piante (IPP), Consiglio Nazionale delle Ricerche (CNR),
via Madonna del Piano 10, I-50019 Sesto Fiorentino, FI, Italy
e-mail: [email protected]
M. Staudt
Centre d’Ecologie Fonctionnelle et Evolutive, UMR 5175, 1919 Route de Mende,
34293 Montpellier cedex 5, France
J. Peñuelas
CSIC, Global Ecology Unit, CREAF-CEAB-UAB, Cerdanyola del Valles, 08193 Catalonia, Spain
CREAF, Cerdanyola del Valles, 08193 Catalonia, Spain

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 1
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 1,
© Springer ScienceCBusiness Media Dordrecht 2013
2 S. Fineschi et al.

1.1 Introduction: The Spectrum of Volatile Organic

Compounds in Plants and the Importance
of Constitutive Volatile Isoprenoids

The emission of biogenic volatile organic compounds (BVOCs) by plants was first
reported in the 1950s and 1960s (see the Preface; Rasmussen and Went 1965;
Sanadze and Kalandaze 1966) and has been studied since by plant biologists and
atmospheric chemists. The amount of hydrocarbons emitted into the atmosphere by
plants as BVOCs far exceeds the levels from human activity (Sharkey et al. 2008;
Peñuelas and Staudt 2010). The large scale of emission, the reactive properties, and
the large capacity of many organisms to sense them render several of these products
of secondary plant metabolism as particularly important for the chemical and phys-
ical properties of the atmosphere. BVOCs also play a critical role in the interaction
between the biosphere and atmosphere, in addition to their role in communication
amongst organisms (Peñuelas et al. 2009a, b; Dicke and Loreto 2010).
Plants produce several secondary metabolites, which can be classified in three
main groups: isoprenoids, phenolics, and nitrogenous compounds. Volatile iso-
prenoids (isoprene, monoterpenes, and some sesquiterpenes) can be either directly
emitted, as is always the case for isoprene, or stored in resin ducts or in glandular
trichomes for later release, especially upon mechanical stress. All plant organs
can emit isoprenoids; vegetative organs such as leaves and branches (Loreto and
Schnitzler 2010), flowers (Knudsen et al. 1993), and roots can all emit volatiles
(Steeghs et al. 2004). Volatile isoprenoids are generally easily smelled (e.g., the
typical fragrance of flowers, resin, and conifer needles), but isoprene, at its natural
concentration, cannot be detected by human olfactory system. Plants release a very
large amount of carbon as isoprene, estimated to be more than 500 Tg isoprene
year1 (Arneth et al. 2008; Ashworth et al. 2013), more than an order of magnitude
of the amount of isoprene emitted by animals (Sharkey and Loreto 1993). The
emission of volatile isoprenoids is widespread in terrestrial plants, with no apparent
evolutionary or ecological gradient (see below), but high isoprene emitters are
generally only trees or perennials. For the purposes of this book on tree physiology,
we will therefore present evolutionary and ecological considerations related mostly
to isoprene emission.
The reason why plants re-emit up to 10 % of their fixed carbon as isoprenoids
(and a much larger proportion under conditions of stress that substantially inhibit
photosynthesis) remains a debated issue (Sharkey and Yeh 2001; Peñuelas and
Llusià 2004; Vickers et al. 2009) despite the many studies of isoprenoid emission.
Plants very likely invest such high metabolic costs to protect vital organs from both
biotic (insects and pathogens) and abiotic (especially extreme temperatures and
atmospheric pollutants) stresses (Possell and Loreto 2013). Emissions, however,
appear to be “specialized”, because not all isoprenoids function in plant defence.
Isoprene was not believed to have a role in defence against biotic stressors until
two independent reports indicated its capacity to repel insects (Laothawornkitkul
et al. 2008; Loivamäki et al. 2008). On the other hand, the finding that isoprene (and
1 Diversification of Volatile Isoprenoid Emissions from Trees: Evolutionary. . . 3

volatile isoprenoids) protects against abiotic stressors, especially high temperatures

(Sharkey and Singsaas 1995; Peñuelas et al. 2005; Velikova et al. 2011) and
oxidative stress (Loreto and Velikova 2001; Peñuelas and Llusià 2002; Vickers et al.
2009; Possell and Loreto 2013), has repeatedly been confirmed experimentally.
The mechanisms by which isoprene produces its protective effects are also topics
of debate. The ideas that isoprene strengthens thylakoid membranes or scavenges
reactive oxygen species have been discussed (reviewed by Loreto and Schnitzler
2010). Velikova et al. (2012) have recently suggested that membrane stabilisation
may also reduce the production of reactive oxygen and nitrogen species under
conditions of stress, thus, reconciling the two putative roles of isoprene. Despite
the prominent role of isoprene in plant protection against abiotic stressors, only
a relatively few extant plants emit large amounts of isoprene (less than 30 % of
the woody species examined to date), the trait having been lost multiple times
during the course of evolution (Harley et al. 1999, see below). Another intriguing
observation is that plants do not generally emit both isoprene and monoterpenes,
but with exceptions, especially in isoprenoid-storing plants. This feature has often
been observed and was discussed in detail by Harrison et al. (2013). Even within a
single plant species, leaves specialize in emitting either monoterpenes or isoprene
at different times during ontogeny, with an apparent trade-off of carbon between the
different biosynthetic pathways (Brilli et al. 2009).
Constitutive emissions of monoterpenes may have the same role as that of
isoprene. If the mechanism of membrane stabilisation is through lipid solubility
and the capacity to delocalize electrons by conjugated double bonds (Loreto and
Schnitzler 2010), then several monoterpenes can replace isoprene, as is often seen
(e.g., Loreto et al. 2004). Monoterpenes, though, are possibly formed at lower rates,
an issue difficult to resolve due to the frequent accumulation of monoterpenes in
storage pools and despite the fact that twice as much carbon is required to construct
monoterpene skeletons as isoprene skeletons.
Plants that emit monoterpenes and sesquiterpenes, however, accumulate iso-
prenoids in specialized organs in leaves, stems, or trunks and then massively
release them after wounding. The main role of these compounds is thus to act
as powerful deterrents to pathogens and herbivores (Dicke and Baldwin 2010)
and to contribute to the sealing of wounds (Loreto et al. 2008). Constitutive and
induced emissions of volatile isoprenoids may be stimulated by mechanical stresses
and metabolic changes in plants attacked by herbivores. The emitted isoprenoids
can directly attract or deter herbivores (direct defence) or attract parasitoids or
predators of herbivores, thus, eliciting an indirectly induced defence resulting from
the interaction of plants with insects of the third trophic level, i.e., carnivores (Llusià
and Peñuelas 2001; Dicke et al. 2003a, b; Matthes et al. 2010; Dicke and Baldwin
2010; Trowbridge and Stoy 2013). After the first report of the interactions amongst
the three trophic levels (Price et al. 1980), these relationships became an attractive
area for interdisciplinary research involving evolutionary biology, ecology, and plant
physiology. Volatile isoprenoids may also activate mutualisms, e.g., with pollinators
or ants (Farré-Armengol et al. 2013). In insect-plant mutualisms, the partners
involved play different roles. The sedentary partner (the plant) must be easily located
4 S. Fineschi et al.

and must offer rewards (mostly food) to the mobile partner (the insect), who offers
a service but who also has a choice whether or not to visit a particular individual
(Farré-Armengol et al. 2013). This relationship implies that plants have evolved
particular traits contributing to mutualism, whereas insects have not. In fact, the
behavioral repertoire of mutualistic insects is no different from that of their non-
mutualistic relatives. Such an asymmetry in trait evolution is particularly evident in
more generalized insect-plant mutualisms (Bronstein et al. 2006).
Phylogenetic studies suggest that insect-plant mutualisms, despite their eco-
logical importance, may have appeared and disappeared several times throughout
evolutionary history, as has isoprene emission (see below). Pollination and seed
dispersal, where physical factors such as wind, water, or gravity have replaced
insect- and bird-facilitated dispersal (Bronstein et al. 2006), clearly illustrate this
adaptability. Whether changes in BVOC emissions have also played a role in these
shifts is not clear, but is a very interesting question. Notably, in the evolution of
plants, interactions with pollinators and seed dispersers appeared after herbivory
and environmental stresses had already shaped evolution of plants (Farré-Armengol
et al. 2013). More generally, understanding the trade-offs between the costs and
benefits of emitting volatiles is challenging and important from the viewpoint of
evolutionary ecology.

1.2 The Present: Inventories of Volatile Isoprenoids

1.2.1 State-of-the-Art of Emission Inventories

Numerous screening studies have been performed since the 1970s to identify plant
species that emit BVOCs. Beginning in North America but later rapidly expanding
to Europe and other continents, most of these studies have focused on isoprene,
the most abundant BVOC. The results of these species inventories, together with
data on canopy densities and species abundances, were used to calculate BVOC-
emission capacities of landscapes that were further integrated into emission models
to estimate BVOC fluxes at regional, continental, and global scales (Guenther et al.
2006; Ashworth et al. 2013; Guenther 2013).
In addition to their usefulness in estimating emission inventories, data of the
relative abundancies of isoprene-emitting species in regional floras may improve our
understanding of the evolutionary origins and ecological significance of isoprene
emissions. If isoprene production confers significant protection against stresses at
non-negligible metabolic costs, then the patterns of species abundance and species
dominance of isoprene emitters may vary worldwide according to site-specific
conditions. Terrestrial ecosystems, though, differ greatly in age. While communities
of tropical forests may be many millions of years old, those at higher latitudes
emerged from ice-age refugia only a few thousand years ago and they may still be
far from equilibrium. The adaptive significance of isoprene production in younger,
often oligospecific plant communities may differ from that in older, often highly
1 Diversification of Volatile Isoprenoid Emissions from Trees: Evolutionary. . . 5

diversified communities, independent of differences in the occurrence of abiotic

stresses associated with each climate. Furthermore, isoprene emissions may have
initially evolved in only a limited number of plant taxa whose distributions depend
not only on their competitiveness and the potential role of isoprene production
therein, but also on the extent of their past geographical isolation that was governed
by geological events such as continental drift and changes in sea level.
Monson and co-workers (2013) recently combined a large dataset of isoprene
emission with DNA sequence data to reconstruct the taxonomic distribution and
evolutionary history of isoprene emitters throughout the plant kingdom. Their com-
pilation found that all major groups of Gymnosperms (Pinophyta) and Angiosperms
(Magnoliophyta) contain both isoprene-emitting and non-emitting species, with
perhaps a few exceptions. Isoprene emitters seem to be particularly rare in the
subclass Asteridae that consists of modern and highly derived plant clades, many
of which are herbaceous species. Isoprene emitters are also rare in the subclass
Magnoliidae, which is considered a rather ancient clade within the Magnoliophyta
comprising numerous trees and shrubs in tropical and subtropical areas around the
world. Nevertheless, isoprene-emission capacity appears to be associated with the
perennial lifestyle and to be particularly frequent in woody plant species. Isoprene
emission is also present in Pinophyta trees, but emissions appear to be generally low
and limited to a few genera (e.g., Abies).
We examined about 30 emission inventories for the presence of isoprene-
emitting species in the floras form various ecosystems and biomes. From each
inventory, we calculated the fraction of isoprene-emitting species per vegetation
type, defining isoprene emitters as species with reported emission rates clearly
exceeding 1 g g1 h1 (ca. 4 pmol g1 s1 ). We did not include species with
taxonomically assigned emission properties unless we found reliable corroborating
information from other sources. Figure 1.1 displays the mean fraction of emitters per
biome. These results imply a relatively homogenous presence of isoprene emitters
throughout the world’s major biomes, with mean fractions only ranging between
about 20–35 %. The range is, however, considerably larger when comparing
individual screening studies. For example, the fraction of isoprene-emitting species
reported at various locales in China (Fig. 1.2) extends from 10 %, reported by
Klinger et al. (2002) amongst 67 species in the humid forests of the Ailao Moun-
tains, to 46 %, observed by Geron et al. (2006a) amongst 95 species screened in
the tropical Biological Gardens of Xishuangbanna. Worldwide, the average fraction
of isoprene-emitting species across all screening studies considered here is 29 %,
which is close to the values we have extracted from the global BVOC databases
of NCAR and Lancaster University (approximately 32 and 33 %, respectively, for
isoprene-emission potentials >1 g g1 h1 ).
Inferring trends from the geographical variation of the fractions displayed in
Figs. 1.1 and 1.2 is appealing. For example, the relative presence of isoprene
emitters in the floras of tropical and subtropical America tends to decrease from wet
to dry forests, savannas, and desert shrublands. The forests of cold highlands and
boreal regions seem to have fewer isoprene emitters than temperate forests (Figs. 1.1
and 1.2), whereas the flora of suburban areas may be particularly rich in isoprene
6 S. Fineschi et al.

22
25
17
35 27
26 20
21 30

32
36
28
30

Fig. 1.1 Mean fraction of isoprene-emitting plant species in the floras of various ecosystems
around the world (numbers are percentages of isoprene emitters). The colours roughly denote
climate classes. Red: moist tropical; yellow: dry (sub)tropical; light green: dry temperate; dark
green: moist temperate; blue: cold (References: Arey et al. 1995; Bracho-Nunez et al. 2012; Chang
et al. 2012; Geron et al. 2002, 2006a, b; Guenther et al. 1996a, b, 1999; Harley et al. 2003, 2004;
Isebrands et al. 1999; Helmig et al. 1999; Karl et al. 2009; Karlik and Winer 2001; Keenan et al.
2009; Keller and Lerdau 1999; Klinger et al. 1998, 2002; Lerdau and Keller 1997; Lerdau and
Throop 1999; Lindfors et al. 2000; Owen et al. 2001; Parra et al. 2004; Rinne et al. 2009; Singh
et al. 2008; Tsui et al. 2009; Wang et al. 2003; Xiaoshan et al. 2000; BVOC emission databases
of the National Center for Atmospheric Research (http://bvoc.acd.ucar.edu) and the Lancaster
University (http://www.es.lancs.ac.uk/cnhgroup/iso-emissions.pdf))

emitters (Beijing, Hangzhou, and Hong Kong in Fig. 1.2). Many ornamental shrubs
and trees used for private gardening and urban shading are isoprene emitters that
contribute to the atmospheric load of BVOCs in these areas (Niinemets and Peñuelas
2008). Few studies have examined the abundance of isoprene emitters along geo-
graphical, climatic/edaphic, and successional gradients in different continents and
biomes (Klinger et al. 1994, 1998, 2002; Martin and Guenther 1995). The results of
these studies collectively suggest that isoprene emitters, often associated with fast-
growing woody species, are more frequent in transitional, mid-successional forests
than in late “climax” forests that are dominated by evergreen monoterpene-emitting
plants (Harrison et al. 2013).

1.2.2 What Are We Missing in Emission Inventories?

The current species inventories, however, can only be taken as rough estimates of
the true prevalence of isoprene emitters in terrestrial ecosystems. The fraction of
isoprene emitters determined by screening studies at one location can differ by as
1 Diversification of Volatile Isoprenoid Emissions from Trees: Evolutionary. . . 7

Inner Mongolia
Klinger et al. 2002

Changbai Mountain
Klinger et al. 2002
15
Beijing Mountain
Klinger et al. 2002 19
25 Beijing
Xiaoshan et al. 2000
Wang et al. 2003
Kunming 33 29
Klinger et al. 2002
Dinghu Mountain
Klinger et al. 2002
Hangzhou
Chang et al. 2012
Ailao Mountain
33
Klinger et al. 2002
12
10
Hongkong
32
Tsui et al. 2009
Xishuangbanna 46
Klinger et al. 2002
Geron et al. 2006
20 46

Fig. 1.2 Fractions of isoprene-emitting plant species observed in screening studies conducted in
various regions of China (numbers are percentages). Asterisks indicate the approximate locations
of the studies

much as a factor of two (for example, see the inventories of the Xishuangbanna
region in southern China in Fig. 1.2). Inventories are unreliable for several reasons.
First, fewer than 2,000 woody species have been screened for isoprene emission,
and in many inventories, only few species have been measured in particularly
species-rich floras that are composed of thousands of vascular plant species. Second,
screening studies have usually focused on large woody plant species, neglecting
small herbaceous species, probably because the latter are difficult to measure
with current enclosure systems and are considered to contribute less biomass to
the vegetation cover. Thus, if isoprene emission is indeed mostly confined to
woody perennial growth habits, then the real fraction of isoprene-emitting species
present in the world’s floras is likely much lower than those inferred from current
emission inventories (Figs. 1.1 and 1.2) in which annual and biennial plant species
are largely underrepresented. Third, inventories are usually conducted at a single
time of year (generally in summer), but the biosynthesis of constitutive volatile
isoprenoids is under strong ontogenetic, seasonal, and even circadian control (for
overviews, see Loreto and Schnitzler 2010; Niinemets et al. 2010). The situation
is particularly complicated for monoterpenes, amongst which are some compounds
such as the ˇ-ocimenes that are known to be induced by stress and/or to occur
only during a limited time of the year (Staudt et al. 1997, 2000, 2003; Grote et al.
8 S. Fineschi et al.

2013). Fourth, inventories are usually conducted on only a few individuals and
ignore the possibility of intra- and inter-population variability in the emission of
isoprenoids. For example, the emission of volatile isoprenoids by the Mediterranean
oak species Quercus ilex and Q. suber can change qualitatively, and can also be
absent amongst individuals and populations (Loreto et al. 2009; Staudt et al. 2001,
2004, 2008). Fifth, screening studies differ greatly in their applied methods (i.e.,
enclosure systems and analytical facilities), although some efforts have been made
to develop standardized methods and measuring protocols (for an overview, see
Niinemets et al. 2011). Optimal identification and quantification of different classes
of BVOCs require the use of several different sampling and analytical techniques.
Consequently, the same plants, which have been screened for the emission of
isoprene, have often not been screened for the emission of other volatile isoprenoids.
Finally, many plant species previously classified as isoprenoid non-emitters now
emerge as emitters when monitored with more sensitive methods of detection (e.g.,
Peñuelas et al. 2009b).
Our knowledge of the present distribution of BVOC-producing plants in ter-
restrial ecosystems has rapidly progressed in recent decades, but more worldwide
coordinated efforts are needed to obtain realistic data on geographical and taxo-
nomic patterns of volatile isoprenoid production by plants. These systematically
gained data are a prerequisite for understanding and reliable assessment of the past
and future evolution of volatile isoprenoid production.

1.3 The Past: Evolution of Volatile Isoprenoids

1.3.1 Volatile Isoprenoids: “Secondary” or “Primary”

Metabolites?

The evolution of volatile isoprenoids is currently being intensively investigated.

Using the traditional definitions of “secondary metabolites”, volatile isoprenoids
should not be essential for plants. The production of volatile isoprenoids has been
proposed to take advantage of dimethylallyl diphosphate (DMADP) and its isomer,
isopentenyl diphosphate (IDP), both of which are synthesized primarily to produce
essential isoprenoids. Conditions affecting synthesis of the higher isoprenoids will
thus affect the production and emission of volatile isoprenoids (Owen and Peñuelas
2005). According to Peñuelas and Llusià (2004), every BVOC emitted does not
necessarily have a specific role, given that their emission is unavoidable due to their
volatility. In many cases, however, natural selection has taken advantage of this
volatility and conferred upon them important roles in defence or communication
(Peñuelas and Llusià 2004).
Thus, volatile isoprenoids, while traditionally classified as secondary metabo-
lites, are in fact key molecules that require large fractions of fixed carbon (Sharkey
and Yeh 2001) and serve multiple very important physiological and ecological
1 Diversification of Volatile Isoprenoid Emissions from Trees: Evolutionary. . . 9

functions, especially those related to communication and protection against biotic

and abiotic stressors (Loreto and Schnitzler 2010; Possell and Loreto 2013).
Evolution forced by selection can work on available genotypic diversity and modify
the roles of isoprenoids to serve a broad diversity of adaptive roles (Peñuelas and
Llusià 2004), e.g., due to high levels of ozone (Lerdau 2007; Calfapietra et al.
2013) or low levels of CO2 (Way et al. 2011). The human factor may also be an
important driver of evolution by selection, at least after plant domestication. Non-
volatile terpenes, which are also considered as “secondary metabolites”, are often
not “secondary” for humans. For example, morphine and codeine, two alkaloids
produced by Papaver somniferum, link “ecology, evolution, and human affairs” very
well, as noted by Theis and Lerdau (2003). In other cases, intraspecific differences
in the emission of volatile isoprenoids may have no evolutionary significance per
se, but are associated with suitable traits for cultivation. A similar hypothesis has
been invoked by Loreto et al. (2009) to explain why Portuguese cork oaks that have
been selected for the quality of cork also emit a blend of constitutive monoterpenes
characterized by a much higher emission of limonene compared to the blend emitted
elsewhere in the range of this plant species.
The “raison d’être of secondary plant substances” has been investigated for more
than 50 years (Fraenkel 1959) and is still being discussed (Berenbaum and Zangerl
2008), but we now believe that plants synthesize their secondary compounds,
including volatile isoprenoids, to fulfill specific needs. Just as floral scents and
pigments were selected to attract pollinators, or toxic non-volatile secondary
compounds to repel herbivores and pathogens, evolution may have selected the
biosynthesis of volatiles emitted from leaves. Thus, research into the ecological and
physiological roles of isoprenoids have led us to question the firm meaning of the
term “secondary plant compound”. In fact, secondary and primary roles are often
indistinguishable with regard to enhancing plant fitness.

1.3.2 Loss and Gain of Isoprene Emission Capacity:

When and Why?

Past inventories suggest that the emission of isoprene and monoterpenes is scattered
across plant divisions (Harley et al. 1999; Kesselmeier and Staudt 1999; Bagnoli
et al. 2012). In fact, isoprene emission occurs in Bryophyta (mosses), Pteridophyta
(ferns), Pinophyta (conifers), and Magnoliophyta (angiosperms), both monocots
and dicots, independent of phylogeny. This evidence has suggested, as commented
above, that the capacity for isoprene emission may have been gained and lost
several times during the evolutionary history of plants (Sharkey et al. 2008;
Monson et al. 2013).
If isoprenoid emission is under evolutionary control, then an evolutionary per-
spective can help us to understand why only some plants emit isoprene or monoter-
penes. According to Harley et al. (1999), the enzyme responsible for isoprene
10 S. Fineschi et al.

biosynthesis, isoprene synthase (IspS), may have evolved several times indepen-
dently, but Hanson et al. (1999) proposed that the trait of isoprene emission evolved
only once and was lost many times, accounting for its heterogeneous distribution
among taxa. More recently, Lerdau and Gray (2003) suggested an independent
origin of isoprene emission in Pinophyta and Magnoliophyta, with multiple losses of
the trait accounting for the distribution of isoprene emission within Magnoliophyta.
In fact, recurrent losses and neo-formations of isoprenoid synthase genes have
been indicated by several independent studies. For example, Welter et al. (2012)
observed that the production of isoprenoids by Q. afares, an oak species resulting
from an ancient hybridization between an isoprene-emitting and a monoterpene-
emitting oak, has strongly diverged from its parental species, including the complete
suppression of isoprene production. The most recent examination of phylogenetic
relationships (Monson et al. 2013) has: (i) confirmed the “multiple gain – multiple
loss” model of isoprene evolution in both Pteridophyta (ferns) and Magnoliophyta,
(ii) suggested that isoprene synthase genes arose frequently from mutations of
terpene synthase genes (incidentally, this origin is reminiscent of the finding that
mono- and sesquiterpene synthases in Pinophyta and Magnoliophyta have evolved
from an ancestral diterpene synthase (Chen et al. 2011)), and (iii) indicated that
isoprene production has been widely lost, and retained only under a few conditions.
Sharkey et al. (2013), basing their analysis on the chronology of rosid evolution,
suggest that isoprene-emitters appeared during the Cretaceous, and that the trait was
subsequently lost multiple times until present. Monson et al. (2013) hypothesize that
the loss of isoprene may have had different causes depending on whether isoprene
emission was an adaptive or neutral trait. The idea that the trait is adaptive is more
appealing, because the trait would be lost whenever the cost of isoprene biosynthesis
exceeded its adaptive value. The frequency of loss during evolution suggests only a
narrow range of conditions in which isoprene has adaptive value.
If isoprene has adaptive significance, what are the conditions under which the
trait is retained?
(a) Isoprene emission is particularly widespread in hygrophytes. Hanson et al.
(1999) and Vickers et al. (2009) therefore suggested that isoprene evolution
could be beneficial when plants are under more recurrent and stronger oxidative
stress in terrestrial than in aquatic environments. About 80 % of European
hygrophytes emit isoprene, a figure significantly higher than in xerophytes
(Loreto et al. unpublished data). However, it may be possible that hygrophytes
diversified less than other plant functional types in which the trait was lost more
often. Even resurrection plants that can survive in extremely dry environments
have recently been found to emit isoprene (Beckett et al. 2012).
(b) Isoprene emission is a characteristic of perennial plants. This restriction may
suggest a relationship with the phloem-loading type, being active phloem
loading widespread in trees, and associated with lower concentration of
leaf non-structural carbohydrates, which are then made available for growth
(Turgeon 2010). However, it was also suggested that high sugar content in
the mesophyll is needed to provide substrate for isoprene (Logan et al. 2000;
1 Diversification of Volatile Isoprenoid Emissions from Trees: Evolutionary. . . 11

Kerstiens and Possell 2001). A relationship between isoprene emission and

sugar accumulation has not yet been demonstrated.
(c) Isoprene emission is a characteristic of fast-growing perennial plants. Again,
this restriction postulates a direct link between the rapid metabolism of carbon
and the need to produce isoprene. In both cases, the benefit of producing
isoprene is unclear, unless plants need to release extra carbon and energy when
the machinery of carbon metabolism is maximally active, an idea reminiscent of
the “overflow valve” hypothesis (Logan et al. 2000) and consistent with the idea
that unstressed plants produce more isoprene because the carbon is not needed
for structural or defensive compounds (the “opportunistic hypothesis”, Owen
and Peñuelas 2005). Not all strong isoprene emitters, though, are fast-growing
(e.g., some deciduous oaks are slow-growing), and even amongst fast-growing
plants, no clear relationship has been found between primary metabolism
(photosynthesis) and isoprene emission (Guidolotti et al. 2011). Nevertheless,
there is evidence of prevalence of isoprene emission among early-successional
trees that typically grow in high light environments and have greater growth
rates than late-successional species (Niinemets and Valladares 2006; Valladares
and Niinemets 2008; Harrison et al. 2013).
(d) Isoprene emission is stimulated by low concentrations of CO2 . This character-
istic has been interpreted as evidence that isoprene may have had an adaptive
advantage during those epochs in geological history when atmospheric CO2
concentration was low (Way et al. 2011). Epochs characterized by low levels
of CO2 , however, were also cold, whereas isoprene emission is stimulated by
high temperatures, and its function is associated with foliar thermotolerance
(Sharkey and Yeh 2001). The evolution of the isoprene trait in epochs with
low concentrations of CO2 would be difficult to explain within this scenario.
However, it may be argued that even in cold epochs, parts of the globe expe-
rienced a temperate climate. On more physiological grounds, isoprene might
be needed when photosynthesis is constrained by low CO2 , and associated
with environmental stresses (Possell and Loreto 2013). This would explain why
isoprene evolved under low CO2 but does not explain why isoprene is today a
widespread trait in fast-growing trees (see (c) above).

1.4 The Future: Impacts of Climate Change and Ecological

Constraints on the Diversification and Emission
of Volatile Organic Compounds

The pace and extent of climate change will affect isoprenoid emissions. Temperature
and CO2 are key climate change factors controlling isoprene emissions. Light,
water availability, and pollution, are also likely going to interfere with isoprene
production and emission capacity by plants (Calfapietra et al. 2013; Holopainen
et al. 2013). Because of the well-known dependence of synthesis and volatilization
12 S. Fineschi et al.

of volatile isoprenoids on temperature (Niinemets et al. 2004), any further increase

in temperature (as foreseen by IPCC 2007) will cause an increase in isoprenoid
emission by plants, thus, inducing (alluding to monoterpene odor) ‘a more fragrant
world’ (Peñuelas and Staudt 2010). Indeed, rising temperatures since the late
nineteenth century may have already induced higher emissions worldwide.
Higher temperatures are mainly due to the accumulation of greenhouse gases,
primarily CO2 . Rising levels of CO2 have a negative effect on isoprene, docu-
mented by many reports since Sanadze (1964) and reviewed elsewhere (Loreto
and Schnitzler 2010; but see Sun et al. 2012; Calfapietra et al. 2013; Monson
2013). The impacts of simultaneous increases in temperature and CO2 levels on
isoprene emission may thus virtually cancel out, with a residual stimulatory effect
due to higher CO2 -driven biomass production and leaf area index (Arneth et al.
2008). A similar conclusion was reached by Heald et al. (2009) using a different
algorithm, and therefore, this scenario is rather probable. Rising levels of CO2
appear not to have a similar inhibitory effect on monoterpenes, so the future impact
of rising CO2 levels on these compounds cannot be predicted using the same
parameterization as for isoprene (Arneth et al. 2008). Although clearly there are
more data needed on [CO2 ] effects on monoterpene emissions. More importantly,
volatile isoprenoid emissions can be transiently enhanced by high temperatures that
are repeatedly occurring worldwide (Rennenberg et al. 2006). In the absence of a
concurrent increase in CO2 levels, this effect appears to be much more dramatic and
deserves thorough analysis, especially if the frequency of episodic extremely high
temperatures will indeed increase in the future.
In areas where rising temperatures and enhanced evapotranspiration will reduce
water availability, isoprenoid emission may also be affected by drought. Stress from
drought appears to have a complex impact on isoprenoids, but isoprene biosynthesis
is generally resistant to drought and increases the metabolic cost of isoprenoids
when carbon acquisition by photosynthesis is inhibited (Loreto and Schnitzler 2010;
Possell and Loreto 2013). Interestingly, the dependence of isoprenoid emission on
temperature changes in leaves severely stressed by, or recovering from drought,
suggesting that a further feedback operates on the main driver of isoprene emission;
this can be a possible additional factor reducing isoprenoid emission in a warmer
world (Bertin and Staudt 1996; Fortunati et al. 2008).
Changes in incident light, for example through an increase in the atmospheric
load of aerosols that increase the fraction of diffuse light (Mercado et al. 2009), may
have large impacts on the future release of isoprenoids from vegetation (Kulmala
et al. 2013). Foliar isoprenoid emissions normally increase with increasing incident
radiation. Combined with extreme temperatures or drought, though, strong radiation
amplifies oxidative stress inside leaves, which can efficiently reduce the total
amount, alter the composition, and modify the thermal responses of isoprenoid
emissions (Staudt and Lhoutellier 2011). Such unaccounted interactive effects of
environmental drivers on isoprenoid emissions may explain some of the observed
variability in the response of emissions to single factors. For example, monoterpene
emissions from Mediterranean oaks appear to be much more resistant to drought
when studied in controlled mono-factorial laboratory and greenhouse conditions
1 Diversification of Volatile Isoprenoid Emissions from Trees: Evolutionary. . . 13

(Bertin and Staudt 1996; Staudt et al. 2008) than under field conditions (Staudt
et al. 2002; Lavoir et al. 2009). More studies of the interactions caused by stress are
certainly needed to reliably predict the future evolution of isoprenoid emissions.
The impact of pollutants on isoprenoid emission is controversial. Air pollution
appears to stimulate or maintain isoprenoid biosynthesis and emission, although
not always (Peñuelas et al. 1999; Loreto et al. 2004; Calfapietra et al. 2009, 2013;
Holopainen et al. 2013). Calfapietra et al. (2009) hypothesized a hormetic response,
with pollution stimulating isoprenoid emission until the inhibition of photosynthesis
no longer allows for a sufficient production of isoprenoid substrates. Again, as in the
case of drought, pollutants will increase the costs of carbon and energy of isoprenoid
formation. Why is this? We suggest that this results from the circumstance that
isoprenoids interact with reactive oxidative species, thereby reducing membrane
damage and stabilising photosynthesis (Loreto and Schnitzler 2010). Enhanced
isoprenoid emission may thus reflect the activation of a defensive system by plants
against stress.
If volatile isoprenoids protect plants against high temperatures and oxidative
stresses, then high emitters should be fitter in a warmer, drier and more polluted
world. In fact, because most natural ecosystems are composed of both emitters and
non-emitters of volatile isoprenoids, emitters may ultimately be selectively favored
by evolution (Lerdau 2007). On the other hand, because most isoprene emitters are
woody species, man- and climate-driven conversion of forested areas to cropland,
savanna, arid shrubland, and grassland may dramatically change the pattern of
emission and reduce the emission of isoprene (Wiedinmyer et al. 2006). Moreover,
because many isoprene emitters are deciduous, and warming will favor evergreen
plants, isoprene emitters may be disfavored, e.g., in boreal areas that will experience
milder temperatures. Another important alteration in the agro-ecosystems may
come from changes in land use involving intensive cultivation of fast-growth
plantations. Most of these fast-growing plants including poplars (Populus spp.),
willows (Salix spp.), eucalypts (Eucalyptus spp.), oil palm (Elaeis guineensis),
and reed (Phragmites australis), are all strong isoprene emitters, with eucalypts
also emitting monoterpenes from storage organs. An expansion of plantations of
these species worldwide is thus expected to vastly increase the biogenic emission
of isoprene. For example, very large areas of rainforest in China, Malaysia, and
neighboring countries have been converted to plantations of rubber trees (Hevea
brasiliensis) or oil palms, which release five to ten times more monoterpenes or
isoprene than the natural vegetation (Wang et al. 2007; Hewitt et al. 2009). In
contrast, biofuel-producing C4 plants do not emit isoprenoids in large amounts
(Graus et al. 2011), though low emissions of isoprene and monoterpenes have
been reported in switchgrass (Panicum virgatum) (Eller et al. 2012) and corn
(Zea mays) may emit several isoprenoids, predominantly sesquiterpenes, when and
after enduring pest attacks (Ton et al. 2007). The massive cultivation of C4 biofuel
plants in the future may thus not affect or may decrease regional fluxes of isoprene.
These crops, though, release significant amounts of oxygenated BVOCs of low
molecular weight, such as methanol, accounting for several grams per liter of biofuel
14 S. Fineschi et al.

produced. These BVOCs are less reactive than volatile isoprenoids and are therefore
less efficient as drivers of atmospheric chemistry.
As is evident from all of the above, the way the emissions of volatile isoprenoids
respond to future conditions is still unclear. The overall emission of isoprene may
increase if large forested areas are converted to tree plantations at the current
pace, and if a “greening of the world” would occur because of photosynthesis
stimulation by rising CO2 levels (unlikely given the accompanying warming and
drying; Peñuelas et al. 2011). In any case, the combined effect of climatic and
anthropogenic factors, in particular, the interaction between rising temperature,
CO2 , drought, and pollution, may offset the predicted (Lerdau 2007) evolutionary
shift in favor of isoprene emitters in natural ecosystems. Rather, monoterpene-
emitting plants, which are often evergreens and resistant to drought, with substantial
limitations to CO2 entry in the leaves, may take the highest advantage in terms of
photosynthesis and growth from rising CO2 levels (Niinemets et al. 2011). Because
monoterpene emission is stimulated by rising temperatures and levels of pollutants,
and is not inhibited by rising levels of CO2 , monoterpene emitters may have the
largest evolutionary advantage, making a warmer world indeed more “fragrant”.

Acknowledgements JP is indebted to the Spanish government projects Consolider Ingenio

MONTES (CSD2008-00040) and CGL2010-17172 and the Catalan government project
SGR2009-458.

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Chapter 2
BVOC-Mediated Plant-Herbivore Interactions

Amy M. Trowbridge and Paul C. Stoy

Abstract Plants release unique blends of biogenic volatile organic compounds

(BVOCs) into the atmosphere, part of a silent language used to communicate with
other organisms in their community. Within this high traffic chemical environment,
plants and insects, among other organisms, are receiving, processing, modifying,
and responding to information conveyed through varying suites of molecules. Be-
cause plants and insects are part of an integrative complex of food web relationships,
one common topic of conversation is defence. Plants maintain a baseline level of
BVOC emissions as a bottom-up constitutive defence, emitting compounds that
act as repellents or deterrents to feeding and/or egg deposition by herbivores. Due
to the autonomy of their attackers, plants can also employ an indirect top-down
defence strategy, releasing induced volatiles in response to feeding that attract the
natural enemies of their herbivore attackers, such as predators and parasitoids. Both
bottom-up and top-down BVOC-mediated strategies have important consequences
for herbivore preference, performance, and survival with even broader ecological
and evolutionary consequences for tritrophic interactions. In this chapter we discuss
how constitutive BVOCs mediate aspects of plant defence within a hierarchical
spatiotemporal framework. Next we bring to light some of the most recent research
on oviposition- and herbivore-induced BVOC synthesis and subsequent effects on
the recruitment of natural enemies. We follow up by discussing the ecological effects
of induced BVOCs in the context of multiple herbivores, expression from various
plant organs, time-lags associated with BVOC induction, and heterogeneity within
the infochemical environment. The critical feature of insect learning is described
and we highlight some of the major evolutionary implications of BVOC-mediated
plant defence syndromes that rely on the unique timing of events at the biochemical,
atmospheric, organismal, and community scales.

A.M. Trowbridge () • P.C. Stoy

Department of Land Resources and Environmental Sciences, Montana State
University, Bozeman, MT 59717, USA
e-mail: [email protected]

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 21
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 2,
© Springer ScienceCBusiness Media Dordrecht 2013
22 A.M. Trowbridge and P.C. Stoy

2.1 Introduction

Organisms are in continuous communication with each other to facilitate survival,

defence, cooperation, and social connections. The study of the ability of organisms
to interpret and communicate information is known as biosemiotics (von Uexküll
1926) and extends far beyond the signals that human are most adept at perceiving.
We are constantly in the midst of countless mutualistic, antagonistic, and uninfor-
mative dialogs that are carried out in a chemical language that developed long before
humans walked the Earth. The terrestrial fossil record provides evidence of volatile
chemical signalling at the beginnings of the evolutionary arms race between species
via structures such as plant essential oil glands (Fahn 2002; Krings et al. 2002)
and olfactory appendages (Strausfeld and Hildebrand 1999; Labandeira 2002). The
ability of plants to communicate with plants and other organisms via volatile organic
compounds (BVOCs) is fascinating, and influences important evolutionary and
ecological processes that we are only beginning to understand.

Definitions
Allelopathy: Biogenic phenomenon where secondary chemicals produced by one
organism affect the development, growth, survival, and reproduction of other
organisms.
Constitutive defence: A plant defence strategy that is always expressed in the plant.
Direct plant defence: Plant traits that negatively influence the physiology or
behavior of herbivores.
Indirect plant defence: Plant traits that enhance the efficacy of the natural enemies
of herbivores, such as herbivore-induced BVOCs.
Induced response: Change in a plant following stress or damage.
Induced resistance: An induced response that reduces herbivore survival, repro-
duction, or preference for the plant of interest, but may not necessarily benefit
the plant.
Induced defence: A response that decreases the negative fitness consequences of
attacks on plants, but may not necessarily affect herbivores.
Infochemical: A chemical that conveys information between two organisms or
individuals resulting in a physiological or behavioral response in the receiver
that is adaptive to one or both parties.
Tritrophic interactions: Relationship between a plant, an herbivore, and the natural
enemies of the herbivore, mediated by plant chemistry and/or BVOCs.

Plants emit more than 30,000 different BVOCs including terpenoids, green
leaf volatiles, phenylpropanoids, benzenoids, and methyl esters of plant hormones
(i.e., methyl jasmonate and methyl salicylate). These BVOCs mediate plant-plant,
plant-insect, and multitrophic interactions and play critical roles in plant defence
(Yuan et al. 2009; Fineschi et al. 2013). Effective defence is critical for plant survival
and reproduction as plants are subject to constant attack from pathogens, fungi,
insects, mammalian herbivores, and other biotic agents. To combat herbivory by
2 BVOC-Mediated Plant-Herbivore Interactions 23

Fig. 2.1 Scheme highlighting the role of biogenic volatile organic compounds (BVOCs) in
mediating interactions between trees and herbivores. Plants constitutively synthesize and release
BVOCs from leaves, stems, and roots, as demonstrated on the left hand side of the figure.
Constitutive BVOC emissions from different organs within a tree and trees within a forest (blue) are
transported to the atmosphere (black lines) where they are mixed by turbulence. Different mixtures
and concentrations of BVOCs are present in different plant parts, as indicated by coloration.
Constitutive BVOC emissions are also mixed with BVOCs whose synthesis and/or emission was
induced by herbivores, as demonstrated on the right-hand side of the figure, resulting in the total
BVOC signal in red. Herbivores perceive and respond to BVOC concentrations within the plant
(red dashed lines), and their feeding activities may induce plants to produce and release a different
combination of BVOCs locally, or in other parts of the plant as demonstrated by the dashed white
lines. Other organisms perceive and respond to BVOCs in their environment (or Umwelt following
von Uexküll 1926), as represented by dashed black lines. The multitrophic interactions that are
mediated by BVOCs, including herbivore activity and parasitoid oviposition behavior, are central
components of forest population, community and ecosystem ecology

heterotrophic organisms that span multiple taxa and developmental stages, plants
employ a diverse arsenal of chemical defence mechanisms that include BVOCs.
Thus, BVOCs are an integral part of a comprehensive bottom-up and top-down plant
defence strategy because they enable plants to resist herbivory within and beyond
the leaf, stem, and root (Fig. 2.1).
This chapter focuses on the role BVOCs play in interactions between plants and
insects. We place particular emphasis on spatial and temporal aspects of both consti-
tutive and insect-induced BVOC dynamics to highlight the role played by BVOCs in
tritrophic ecological interactions. We also discuss above- and belowground factors
24 A.M. Trowbridge and P.C. Stoy

that control BVOC emissions, and the influence of the resulting emissions on
tritrophic interactions associated with both roots and leaves. Our focus here is on
biotic interactions in natural unpolluted environments, and we refer the reader to the
chapter of Holopainen et al. (2013) in this book for a discussion of the modification
of biotic interactions in polluted atmospheres and under global change. We further
refer the reader to Peñuelas and Llusià (2003), Loreto and Schnitzler (2010), and
Possell and Loreto (2013) and Calfapietra et al. (2013) in this book about the role
of abiotic stresses, including climate change, in altering BVOCs, and subsequent
consequences for ecological signalling. We conclude with a brief overview of the
evolutionary implications of BVOCs in the context of insect learning in complex
environments.

2.2 Constitutive BVOCs

Constitutive BVOCs serve as constant barriers to herbivore attack by deterring

colonization through their antixenotic function and inhibiting growth, reproduction,
development, and/or survival through their antibiotic function (Paiva 2000; Walling
2000). We refer to these characteristics as ‘bottom-up’ defences as they directly
impact herbivore performance. In a natural setting, both the rate at which volatiles
are released and their spatial and temporal persistence in the environment determine
their relevance in ecological signalling. However, complex feedbacks exist between
the phylogenetic constraints on biosynthesis, environmental controls on emissions,
and compound-specific physico-chemical responses to the environment. As a result,
the spatial and temporal heterogeneity of BVOC emission rates is often pronounced,
with important implications for multispecies interactions. We discuss consequences
of BVOC emissions at the leaf level and then describe controls on and consequences
of BOC emissions at the organ, whole plant, and community scale.

2.2.1 Role of Constitutive BVOCs in Defence

and Host Selection

Trees maintain a baseline level of volatile metabolites that are released from the
leaf upon production, or some time after production from storage sites (e.g., resin
canals) (Paré and Tumlinson 1999). Within the last decade, an emphasis has been
placed on understanding the role of constitutive vegetative BVOCs in deterring
herbivory. Unfortunately we still lack a mechanistic understanding of how BVOCs
released from plants directly repel herbivores and/or inhibit feeding. One possibility
is that constitutive emissions are directly toxic to impending attackers, potentially
affecting physiological and neurological processes by influencing gene expression
and/or interfering with the macronutrient digestion. However, studies have yet to
2 BVOC-Mediated Plant-Herbivore Interactions 25

demonstrate how relatively low-emission rates, typically on the scale of nano-

or micrograms per hour per gram of leaf tissue, elicit toxic physiological effects
(Unsicker et al. 2009).
Herbivores must discriminate between true ecological signals that might increase
their fitness, and background noise resulting from a highly variable infochemical
environment. Most phytophagous insects are specialist feeders that utilise a limited
number of species as resources, and chemical cues are critical in selecting suitable
hosts. It is not in the best interest of a plant to attract herbivores; however,
specialists are capable of exploiting the characteristic volatiles emitted by their
hosts to gain information on quality, susceptibility, and natural enemy status.
Reddy and Guerrero (2004) give two examples of how insects exploit BVOCs with
different pheromone-mediated implications for plant resistance. Some insects can
use constitutive BVOCs for their own defence. For instance, green leaf volatiles
(GLVs) released from non-host tree species serve as repellents in host selection
and inhibit the pheromone response of several bark beetles. Other insects can
use constitutive BVOCs to their advantage in finding mates. When male Ips
and Dendroctonus spp. are exposed to the volatile myrcene, they modify the
compound, which results in the production of oxygenated pheromones in their
hindgut. However, predators and parasitoids attacking adults and eggs have learned
to eavesdrop on these pheromone signals to locate prey more effectively (Stowe
et al. 1995). As a consequence, constitutive BVOC signals that are attractive to
specialists can be intercepted and used by the herbivore’s natural enemies as a
type of indirect defence. Thus, constitutive and induced defence strategies should
be assessed in an integrative fashion to fully appreciate the ecological role played
by BVOCs.

2.2.2 Spatiotemporal Patterns

2.2.2.1 Leaf-Level Responses

BVOC emissions exhibit significant variations across space and time at the scales of
leaves, organs, whole plants, and ecosystems. At the leaf level, rates of constitutive
BVOC production and emission are sensitive to a number of abiotic factors, includ-
ing light and temperature as discussed by Niinemets et al. (2004) and other chapters
in this volume. These factors can interact with ontogenetic changes in emissions
and result in variable emission rates from leaves close in proximity. For instance,
constitutive monoterpene (C10 ) emissions can be detected from young hybrid poplar
leaves (Populus deltoides x Populus nigra). However, mature leaves on the same
tree were found to emit only isoprene (C5 ) and at a rate of about five times greater
than monoterpenes released from young leaves (Brilli et al. 2009), suggesting
that the amount of carbon invested towards isoprenoid biosynthesis changes with
leaf ontogeny. Differences in BVOC quality and quantity between leaves have
26 A.M. Trowbridge and P.C. Stoy

important consequences for herbivore host selection, and these differences can
result from the presence of specialized structures and/or the vascular connections
within the tree. Even subtle spatial variation can be critical for specialists that
have learned to associate proximate volatile signals with host quality. For example,
BVOC emissions tend to be significantly higher in sunlit leaves when compared
to shaded leaves on the same plant and/or branch, due largely to an increase in
light and temperature and higher rates of physiological activity (Harley et al. 1996).
While this increase in emissions may have consequences for herbivore preference,
it is also possible that BVOCs serve multiple ecological functions simultaneously
(e.g., protecting leaves from photodamage at high temperature and light conditions)
(Peñuelas and Llusià 2002) with evolutionary implications via multiple selection
pressures. Furthermore, some plant BVOCs with low vapour pressures tend to
condense on leaf surface as a function of leaf temperature and position in the canopy
and may serve as defence agents against herbivores and pathogens (Holopainen and
Gershenzon 2010), but it is unknown if this mode of BVOC function represents an
effective defence.

2.2.2.2 BVOC Variation Between Vegetation and Flowers

The suite of volatiles released by healthy trees can also vary spatially within a
plant among organs including leaves, stems, flowers, and roots (Fineschi and Loreto
2012; Mumm and Hilker 2006; Takabayashi et al. 1994). The survival of a plant is
determined by investment in reproduction (e.g., flowers, nectar) and growth (e.g.,
vegetative biomass). Thus, allocation of resources to defence may come at the
expense of investment in reproductive tissues and affect mutualistic relationships,
pollination events, and fitness (Mothershead and Marquis 2000). For instance, floral
and vegetative BVOC emissions are differentially attractive and repellent to species
that specifically feed on those organs, yet BVOCs from both plant parts are likely
to be mixed in the atmosphere before their perception by an insect. Thus, chemical
information from other plant organs may confound perception and host identifi-
cation, particularly in response to damage (see Induced Responses). Because both
herbivores and pollinators are selective agents on floral chemistry and emissions, it
is critical to understand the degree to which defensive vegetative BVOC production
alters nectar and pollen quality and thereby affects fitness. Furthermore, compound
type and emission rate can vary substantially between organs as a function of the
predictability of attack on various plant parts (see optimal defence theory, Rhoades
(1979); Zangerl and Rutledge (1996)) and ecophysiological constraints (e.g., tissue-
specific biosynthesis or allocation, Niinemets et al. 2004). While many BVOCs
appear to be exclusively produced in either flowers or leaves, compounds can also
be passively transported into the nectar and/or pollen and subsequently released
into the atmosphere. Future studies should focus on plant allocation and tissue type
to fully understand the dynamics of plant-pollinator-herbivore-interactions (Kessler
and Halitschke 2009).
2 BVOC-Mediated Plant-Herbivore Interactions 27

2.2.2.3 Belowground BVOC-Mediated Interactions

While most studies focus on BVOC-mediated interactions between aboveground

plant parts and herbivores, plants must also defend themselves against soil-dwelling
herbivores including rodents, insects, and nematodes, all of which play key roles
in terrestrial ecosystem community structure. Due to the limited mobility of soil
organisms and the low transport rates of root compounds, interactions in the
soil occur over significantly smaller spatial scales than aboveground interactions
(van der Putten et al. 2001). Despite the difference in characteristic spatial scales
of influence, the role of BVOCs might be more important for belowground
communities where visual cues are lacking and with soil-dwelling herbivores often
exhibiting poor eyesight (Rasmann et al. 2005; van Dam and Heil 2011). A number
of compound classes emitted from aboveground organs have been shown to mediate
plant-herbivore interactions belowground, but in different compositions (Wenke
et al. 2010). Nonetheless, BVOCs can directly or indirectly influence belowground
communities, support symbioses, combat competitive plant species, and defend
against pathogenic fungi, bacteria, and herbivores (Nardi et al. 2000).
Herbivore host choice for oviposition is critical for the next successful generation
of soil dwelling herbivores. Adults of the large pine weevil (Hylobius abietis)
are attracted to suitable hosts in a dose-dependent manner, depending on BVOC
concentrations released from conifer roots (Nordlander et al. 1986). BVOC compo-
sition is also important; larvae of the forest cockchafer (Melolontha hippocastani)
exhibited a noted preference for monoterpenes released from carrots over the fatty
acid derivatives emitted from oak roots (Weissteiner and Schütz 2006). Despite
being separated in space, roots, shoots, leaves, and flowers are connected and so
are their resources, metabolic activities, and defences. Belowground organisms can
induce defence responses aboveground and vice versa, the cost: benefit ratio of
these induced responses and their consequences are discussed in more detail in the
Sect. 2.3: Induced BVOCs.

2.2.2.4 BVOC Variability Between Plants

At the whole-plant level, BVOC emission rates are dependent on a number of

environmental and biotic factors, including developmental stage as well as plant
species, genotype, and age (see Dicke 1999; Paré and Tumlinson 1999). Consti-
tutive plant defences are expressed differentially with ontogeny, and while BVOC
emissions have been shown to decrease in mature cultivated herbs (Cole 1980),
information about their ontogenetic patterns in mature trees and seedlings remains
largely unknown (Boege et al. 2011). Studies focused on ontogenetic changes in
tree BVOC emissions, foliar chemistry, and predator/parasitoid foraging dynamics
may offer mechanistic insight into tritrophic patterns observed in the field. In
addition to age, specific chemotypes and plant varieties have differential growth and
resistance properties (Staudt et al. 2001). For example, mango (Mangifera indica)
tree cultivars that are the most susceptible to mango gall flies (Procontarinia spp.)
28 A.M. Trowbridge and P.C. Stoy

emit significantly higher levels of ’-pinene and “-pinene throughout the growing
season; these volatiles are highly attractive to this pest (Augustyn et al. 2010). At
the plant population level, BVOCs emitted by particular genotypes of seedlings
of Pinus pinaster in plantations have been found to be susceptible to a generalist
phloem-feeding pine weevil (Hylobius abietis). Blanch et al. (2012) showed that
under high nutrient availability, susceptible trees exhibited higher terpene emission
rates, including ’-pinene, an attractant for H. abietis (Moreira et al. 2008), which
could explain the pattern of weevil damage observed in the field (Zas et al. 2008).
Thus, BVOC emission rates must be investigated in the context of plant genetics,
development, and the environment to better-understand herbivore-natural enemy
development, behavior, and natural ecological patterns.

2.2.2.5 Community-Level Variability

Observed spatial variations in foliar BVOC emissions range over several orders of
magnitude at the community level due to changes in species composition and foliar
density (Guenther 1997). Spatial patterns in both the magnitude and composition
of BVOCs that herbivores and their natural enemies perceive are critical for their
behavior, and therefore, for their reproduction and survival. However, the identifi-
cation of specific cues signalling host availability and quality may be influenced by
the background of chemicals present in the habitat from other species, requiring
adaptive and integrative abilities to extract useful search information from the
milieu of chemicals present in the environment (Hilker and McNeil 2008). Drastic
community shifts, via invasive species or changes in climate, can influence plant-
herbivore interactions by altering the proportion of species that produce varying
types and quantities of BVOCs. Depending on the ability of the insect to learn
(see Sect. 2.4, Insect Perception and Learning) and the time required to make
new host-BVOC associations, changing habitats can modify the probability of a
particular plant-herbivore interaction occurring, the intensity of the interaction, and
coevolutionary processes (Agrawal and Fishbein 2006).

2.2.2.6 Variation in Time: Diurnal Cycles of BVOC Release

The effects of time on BVOC emissions extend beyond ontogeny. BVOC emissions
are a product of day length (light), corresponding changes in temperature and
water status, as well as seasonality. Many BVOCs released constitutively from trees
exhibit diurnal cycles, increasing rapidly in the morning with temperature and solar
radiation, peaking in the middle of the day, and decreasing during the afternoon
and evening (Pio et al. 2005; Grabmer et al. 2006). High emissions make plants
more “apparent” to insects, and may determine the employment of other defensive
strategies used by plants to protect against herbivory (Feeny 1976; Rhoades and
Cates 1976). However, “apparency” due to high daytime emissions and their role
in increasing plant vulnerability to herbivores is dependent upon the peak foraging
2 BVOC-Mediated Plant-Herbivore Interactions 29

time of the insect attacker as well as the searching behaviors of their natural enemies.
Furthermore, because vegetative and reproductive tissues exhibit diurnal variability
in emissions, plasticity in volatile production from both types of plant organs can be
critical, as the specificity with which insects choose to visit flowers on fruiting trees
may be the result of the quantitative relationship between the attractant and repellent
components in the blend contributed from leaves (Euler and Baldwin 1996).

2.2.2.7 Variation in Time: Seasonal Trends

Seasonal trends in BVOC emissions have been observed in a number of forest types,
including mixed hardwood (Karl 2003), boreal coniferous (Hakola et al. 2003),
and Mediterranean (Owen et al. 2001; Pio et al. 2005). Similar to emission rates
observed over a shorter time scale, the seasonal release of BVOCs is a function
of changes in light, temperature, and compound-specific physico-chemical controls
(see Niinemets et al. 2004 and Grote et al. 2013 for detailed discussion of controls on
seasonal changes in emissions). For example, within a mixed hardwood forest, Karl
(2003) found, among other patterns, a spring peak for methanol and attributed it to
rapid leaf expansion. While the large amount of methanol released from vegetation
has long been assumed to be a metabolic waste product, studies have shown that
herbivory can also elicit its release, suggesting the potential role of methanol in
mediating plant-insect relationships (Peñuelas et al. 2005). Furthermore, application
of methanol to plants in quantities mimicking herbivore-elicited release affects
bottom-up controls by decreasing plant foliar defences (e.g., trypsin proteinase
inhibitors) and enhancing the performance of the herbivore (von Dahl et al. 2006).
Thus, seasonally-driven BVOC-specific spikes may not only impact plant-insect
signalling, but also force within-plant feedbacks that negatively impact defence
capabilities resulting in higher herbivore pressure at key developmental times of
the year.

2.3 Induced BVOCs

Plants are the primary food source for millions of insect species, each using
unique strategies to obtain nutrients from both above- and belowground tissues.
In contrast to constitutive ‘hard-wired’ plant traits that confer resistance to insect
pests regardless of insect infestation risk, herbivore-challenged plants can exhibit
phenotypic plasticity and can mount active defence responses that are induced
by insect behavior. Trees are thought to have evolved induced defences to save
on allocation costs when pressure from herbivores is low (Heil 2002). When
expressed following herbivory, induced responses can serve as direct defences,
affecting the herbivore through immediate toxicity (i.e., a ‘bottom-up’ defence)
or as an indirect (‘top-down’) defence, affecting the herbivore via recruiting its
30 A.M. Trowbridge and P.C. Stoy

natural enemies (Dicke and Vet 1999). Both induced direct and indirect defences
can alter herbivore behavior and development. BVOCs released immediately after
herbivory consist of preformed volatiles, some resulting from the bursting of storage
structures, and depend on the mode of damage such as wounding, egg deposition,
and herbivore feeding (Walling 2000). Other BVOCs released with feeding are
synthesized de novo and exhibit delayed emissions on time scales of minutes, hours,
days, and potentially seasons. These emissions can also be expressed both locally
and systemically (Paré and Tumlinson 1999). Plant BVOCs are not only mediators
of aboveground plant-insect interactions, but also affect herbivore dynamics in the
soil. In fact, damage by below- and/or aboveground herbivores has been found
to affect pollinators and higher trophic levels, particularly the natural enemies
of herbivores in both root and shoot food webs (see van Dam and Heil (2011)
and references therein). Here we briefly expand upon these topics, with particular
focus on herbivory and oviposition-induced BVOCs and the consequences of the
spatiotemporal dynamics of emissions on above- and belowground defence.

2.3.1 Herbivore and Oviposition-Induced BVOCs:

Induction Depends on Mode of Damage,
Elicitors, and Signal Transduction

The synthesis of novel BVOCs in response to herbivory is not part of a general

syndrome in response to stresses (i.e., drought, ozone, temperature, etc.), but is
a specific response to herbivory with a well-documented defensive role in trophic
interactions (Staudt and Lhoutellier 2007). Some of the earliest studies of induced
host volatiles were performed on trees, (e.g., Populus spp. (Baldwin and Schultz
1983)). With the ability to release hundreds of BVOCs, how do trees release such
specific chemical signals in response to herbivore attack? The quality and quantity
of herbivore-induced BVOCs are dependent on a variety of factors, including the
plant species, plant age, the tissue type being attacked, as well as the herbivore
species, feeding mode, and its developmental stage (De Moraes et al. 1998). The
mode, frequency, and severity of physical damage by herbivores and herbivore-
specific chemical elicitors initiate highly regulated modifications in the plant’s
transcriptional and metabolic processes by activating signalling pathways (Kessler
and Halitschke 2007). While these pathways are well known in herbaceous species,
there exists a gap in our knowledge regarding signals and pathways that induce
resistance in many tree systems. In light of a few recent studies, many assume
similar signal cross-talk and activation in trees as observed in herbaceous plants
(Eyles et al. 2010).
A number of elicitors initiating signal cascades involved in BVOC synthesis have
been isolated and characterized from insect saliva, regurgitants, and oviposition
fluids, and include enzymes, fatty acid-amino acid conjugates, and bruchins (Paré
et al. 2005). Once in contact with plant cells, these elicitors activate signal
2 BVOC-Mediated Plant-Herbivore Interactions 31

transduction pathways (e.g., the octadecanoid (C18 -fatty acids) pathway) that
ultimately lead to gene expression and the synthesis of particular defence-related
BVOCs (for more detail on signal cascades see Kessler and Baldwin (2002)).
Numerous studies in trees have shown that in the absence of wounding, pathways
can also be induced through the application of herbivore oral secretions, elicitors
themselves, or phytohormones such as jasmonic acid (JA), ethylene, and salicylic
acid (SA) (Dicke et al. 1999; Eyles et al. 2010; Van Poecke et al. 2001). The
early steps in the herbivore elicitation process remain to be elucidated as well
as the mechanisms responsible for plant recognition of these herbivore-specific
compounds. Nonetheless, the result of these signal cascades is an herbivore-induced
BVOC blend comprised of tens to hundreds of compounds. While a number of these
compounds are species-specific and actively produced in response to herbivory,
many BVOCs also “leak out” or are released simply due to mechanical damage.
These compounds, known as green leaf volatiles (GLVs), consist of saturated and
unsaturated C6 alcohols, aldehydes, and esters produced by the oxidative breakdown
of membrane lipids (Paré and Tumlinson 1999). Within this complex blend of GLVs
and novel compounds synthesized de novo, however, only a subset of compounds
play a biological role in mediating higher trophic level interactions with herbivores
and natural enemies (see Dicke (2009) and references therein).
Individual signal cascades, as described above, have the ability to serve a variety
of functions. The involvement of several signal cascades in response to specific
forms of herbivory may help explain the specificity of BVOC profiles (Kessler
and Baldwin 2002). As such, plants must be able to not only identify the source
of damage, but also prioritize and tailor the signalling pathway that will mount
the most effective defence strategy (Reymond and Farmer 1998). A rich body of
literature exists regarding induced plant responses to attack by chewing insects and
the subsequent interactions between the two organisms (e.g., Karban and Baldwin
1997). There are also many studies that demonstrate the specific and differential
chemical response of plants to chewing insect species (e.g., De Moraes et al.
1998). However, plants are constituents of complex communities, and as such, are
rarely attacked by a single herbivore. Multiple biotic stressors can significantly
alter herbivore-induced BVOC emissions as concurrent feeding may induce cross-
resistance (Kessler and Halitschke 2007) or competing plant defence pathways,
both of which have important implications for defence and evolution (Rodriguez-
Saona et al. 2005). While far less is known about the induction of BVOCs by
herbivores of feeding guilds that cause less tissue damage (i.e., miners, galls,
and piercing-sucking insects), a study by Delphia et al. (2007) demonstrated
that simultaneous herbivory by insects with different feeding habits significantly
alters BVOC emission and defence strategies. However, ways in which plants
simultaneously integrate responses to multiple herbivores and the ecological and
evolutionary consequences for plant-insect interactions after attack, remains largely
unknown.
Herbivores not only induce changes in plant leaf BVOCs through feeding, but
also through egg deposition. Hilker and Meiners (2002) describe the mechanism
involved in oviposition-induced BVOCs in Scots pine (Pinus sylvestris) and field
32 A.M. Trowbridge and P.C. Stoy

elm (Ulmus minor). In both systems, the adult female wounds the surface of the
leaf and needle just before oviposition. The eggs are then laid into the wounded
tissue along with oviduct secretions that surround the eggs securing them to
the plant. Only when the secretions, containing an elicitor, make it past the
cuticular barrier do leaves release parasitoid attractant BVOCs (Hilker and Meiners
2006). To date, only a few oviposition-associated elicitors have been identified,
including bruchins (Doss 2005) and benzyl cyanide (Fatouros et al. 2008). The
application of jasmonic acid can also elicit the release of BVOCs that attract
the egg parasitoids associated with each species, suggesting the involvement of
the octadecanoid pathway in driving oviposition-induced responses (Hilker and
Meiners 2002; Meiners and Hilker 2000). Similar to herbivory, insect egg deposition
induces plant responses that are specific to both the plant and herbivore species
attacking it, yet whether this specificity is due to species-specific elicitors or a
dosage-dependent response remains unknown for most systems (Hilker and Meiners
2010). While the induced BVOCs produced via feeding and oviposition differ in
composition, both BVOC blends may be perceived by the herbivore and parasitoid
with either negative or positive consequences. For example, herbivore-induced
BVOCs have been shown to deter female herbivores from oviposition in an attempt
to avoid competition (Kessler and Baldwin 2001; De Moraes et al. 2001). Future
work aimed at understanding the interaction of herbivore- and oviposition-induced
signalling pathways, the BVOCs emitted, and consequences for herbivores and
their natural enemies will offer insights into the evolutionary importance of these
compounds.

What is a parasitoid anyway?!?

Parasitoids spend only part of their lifecycle associated with a host. They feed
exclusively in or on the body of another arthropod, eventually killing it. Only a
single host is required for the parasitoid to complete its lifecycle.
Predators kill their prey, usually more than one species, but do not need a host
to complete any part of their lifecycle.
Parasites spend their entire life associated intimately with its host, usually at
the host’s expense, but without causing death.

2.3.2 Induced Volatiles Serve as Direct Plant Defences

Immediately following release, herbivore- and/or oviposition-induced BVOCs carry

a vast array of information through the environment with the potential to directly
influence the behavior of different members of the ecological community. Some
herbivore-induced volatiles have been shown to function as a plant defence by
deterring herbivore feeding and oviposition (Kessler and Baldwin 2001; Laotha-
wornkitkul et al. 2008; De Moraes et al. 2001). For instance, when foraging, starved
adult willow leaf bugs (Plagiodera versicolora) orient towards odors elicited from
2 BVOC-Mediated Plant-Herbivore Interactions 33

willow leaves infested by conspecifics as opposed to intact leaves, perhaps due

to increased quality or lower concentrations of secondary compounds (Yoneya
et al. 2009). Deception is another way in which plants use herbivore-induced
BVOCs to their advantage, such as in the case of the sesquiterpene, (E)-“-farnesene,
which is also an aphid alarm pheromone that signals aphids to stop feeding and
disperse (Bernasconi et al. 1998). Even oviposition-induced BVOCs can affect
the egg laying choice of other female herbivores. To avoid inter- and intraspecific
competition and a site attractive to egg parasitoids, laboratory choice tests showed
adult Xanthogaleruca luteola to prefer BVOCs from field elm (Ulmus minor) leaves
without eggs over those with eggs and/or feeding damage (Hilker and Meiners
2002). In addition to directly resisting the attacking herbivore, induced BVOCs
can also influence herbivores on neighboring plants by priming non-infested plants
to chemically respond faster to future insect attacks. One study showed that rates
of herbivory were lower in black alder (Alnus glutinosa) trees growing close to
damaged conspecifics (Dolch and Tscharntke 2000). This is similar to observations
made by Rhoades (1983), who reported that undamaged Sitka willow trees (Salix
sitchensis) in close proximity to herbivore-infested conspecifics mounted a more
aggressive chemical defence in response to fall webworm larvae (Hyphantria cunea)
than distant controls. If induced BVOCs can directly influence the chemical de-
fences within neighboring trees, it is not surprising that they can also elicit defence
responses in undamaged parts of the same tree. For instance, gypsy moth (Lymantria
dispar) feeding on branches previously exposed to herbivore-induced BVOCs from
nearby damaged branches was reduced by 70% compared with controls (Frost et al.
2007). In addition, extrafloral nectaries have been shown to increase in output when
undamaged leaves are exposed to herbivore-induced BVOCs emitted from damaged
leaves on the same plant, resulting in increased visits from predators (Heil and
Silva Bueno 2007). Despite evidence from experimental observations, we lack an
understanding of how the signals that induce priming are received by plants, which
compounds are biologically active within an herbivore-induced mixture, and the
signalling cascades responsible for indirect BVOC-mediated plant defence.

2.3.3 Induced Volatiles Serve as Indirect Plant Defences

The attraction of the natural enemies of herbivores by damage-induced volatiles

is a well-established phenomenon in many plant species, and probably the first
defence strategy that comes to mind when discussing induced BVOCs. For over
40 years (Green and Ryan 1972), a vast array of herbivore-induced plant BVOCs
has been shown to effectively recruit insects of the third trophic level that prey
upon or parasitize larval herbivores, as well as eggs. By doing so, BVOCs reduce
the preference and/or performance of herbivores, serving as an indirect defence
and an important mediator of tritrophic interactions (Karban and Baldwin 1997).
Nonetheless, because most palatable herbivores are cryptically coloured and well
hidden on the undersides of leaves, the probability of parasitoids effectively finding
their hosts using visual cues and random searches is relatively low. However, the
34 A.M. Trowbridge and P.C. Stoy

suite of compounds released following herbivore-damage is quite sophisticated and

unique, differing in total abundance and composition following attack by different
herbivores (e.g., De Moraes et al. 1998). The species-specific plumes present within
the local environment contain critical host-location information for parasitoids,
which have developed the ability to learn chemical cues associated with the presence
and quality of their specific host (see Sect. 2.4, Insect Perception and Learning).
For instance, some parasitoids are capable of differentiating between parasitized
and unparasitized larval hosts in flight due to the different odor blends induced
by each caterpillar (Fatouros et al. 2005). While herbivore-induced volatile blends
can be quite complex, a number of individual BVOCs involved in attraction of
parasitoids have been identified (e.g., Halitschke et al. 2008; Ibrahim et al. 2005).
However, it is highly unlikely that a parasitoid will be exposed to only one BVOC
in nature, and the context within a BVOC blend is perceived may be important. In
fact, the eulophid egg parasitoid (Chrysonotomyia ruforum), while attracted to the
herbivore-induced release of (E)-“-farnesene, requires the presence of background
non-induced pine odor to locate its sawfly host (Diprion pini) (Mumm and Hilker
2005). While individual herbivore-induced BVOCs may be involved in parasitoid
host location, it is often critical that they are perceived in the context of other BVOCs
so as to distinguish variation in quality and quantity.
Although most available information on BVOC-mediated tritrophic interactions
comes from studies in agricultural or herbaceous species, a number of studies
has also demonstrated the attraction of the natural enemies to herbivore-induced
BVOCs elicited by pests attacking trees, particularly in fruit trees [e.g., apple
(Malus domestica), mango (Mangifera indica), and grapefruit (Citrus paradisi)],
conifers [e.g., Scots pine (Pinus sylvestris) and loblolly pine (Pinus taeda)], as well
as some deciduous species [e.g., elm (Ulmus minor) and black poplar (Populus
nigra)] (see Dicke (1999) and Mumm and Dicke (2010)). For instance, fruit fly
parasitoids (Diachasmimorpha longicaudata: Braconidae) significantly preferred
BVOCs from infested mangoes (with higher BVOC concentrations) and their
extracts (particularly 2-phenylethyl acetate) over healthy and mechanically damaged
fruits, suggesting that parasitoids use induced BVOCs to locate hosts in this system
(Carrasco et al. 2005). In conifers, a dosage-dependent synergistic effect among
pine terpenoids and bark beetle pheromones can attract predators and parasitoids to
their hosts. For instance, the attraction of the predatory beetle Thanasimus dubius
was positively correlated with the concentration of ’-pinene when mixed with the
pheromones of its scolytid prey (Mumm and Hilker 2006). Parasitized herbivores
have also been shown to induce different BVOC blends compared to unparasitized
herbivores, affecting parasitoid choice (Fatouros et al. 2005). However, mutualistic
interactions with herbivores (e.g., aphids and ants) can also alter induced BVOCs
with potential consequences for parasitoid host location (Paris et al. 2011). There
is also evidence that oviposition-induced plant BVOCs successfully recruit egg
parasitoids, such as in the case of Pinus sylvestris and egg deposition by Diprion
pini (Meiners and Hilker 2000). In the deciduous species Ulmus minor, terpenoid
hydrocarbons induced by oviposition of the elm leaf beetle (Xanthogaleruca
luteola) are exploited by the egg parasitoid Oomyzus gallerucae. Despite the work
2 BVOC-Mediated Plant-Herbivore Interactions 35

in these systems, more studies are needed to fill gaps in our knowledge pertaining
to the importance of induced indirect defences employed by trees, particularly in
determining community structure and outbreak dynamics.

2.3.4 Spatiotemporal Aspects of Induced Indirect Defence

Similar to constitutive BVOCs, herbivore-induced BVOCs not only vary over space
and time, but their associated costs to plant fitness vary as well. Most factors
influencing constitutive emissions have similar effects on herbivore-induced volatile
defences as discussed in previous sections here and in other chapters in this
volume. Thus, rather than reiterating these points, we place the spatial and temporal
variability of herbivore-induced BVOCs in an ecological framework by discussing
ecological interactions influenced by the location of induction in a plant and the
timing and patterns associated with the response.

2.3.4.1 Local vs. Systemic Induced Responses

Induced BVOCs that serve a resistant or defensive role for plants can be expressed
locally at the wounding site or systemically via mobile signals and phloem
transport (Turlings and Tumlinson 1992; Heil and Ton 2008). The ability to
respond systemically to herbivory enables a plant to have a larger BVOC response,
potentially serving as long range cues capable of recruiting natural enemies that
forage over spatial scales of metres to kilometres (Puente et al. 2008). Furthermore,
these systemic signals can give parasitoids an initial estimate of patch quality
(e.g., number of hosts in a habitat) to aid in determining whether or not to pursue
hosts in a given area. However, once parasitoids orient themselves within the
general vicinity of their host, the specific blend associated with the herbivore at
the damage site itself becomes more important, and constitutes a reliable indicator
of host location (Cortesero et al. 1997). BVOC communication between branches
or leaves of the same individual could enable faster responses, particularly when
signalling via phloem and xylem is thwarted by limited vascular connections or
distance, for example in larger trees (Dicke 2009). For instance, Frost et al. (2008)
demonstrated that both mechanically-injured and gypsy moth-damaged leaves of
hybrid poplar (Populus deltoides x P. nigra) primed defence responses in undamaged
leaves of the same plant. This “second route” for signal transduction within plants
can provide a relatively large benefit to the emitting plant in lieu of synthesis
costs. Herbivore-infested plants also mediate plant-plant interactions in unattacked
neighboring plants, thus increasing their attractiveness to natural enemies and
decreasing their susceptibility to herbivory (Baldwin et al. 2006). While within-
plant BVOC signalling has gained much interest, interspecific volatile signalling
between plants has remained a topic of debate (Agrawal 2000; Baldwin and Schultz
1983; Bruin and Dicke 2001; Dudareva et al. 2006; Farmer and Ryan 1990).
36 A.M. Trowbridge and P.C. Stoy

2.3.4.2 Spatially Separated but Connected: Above- and Belowground

Induced Responses

Herbivores can attack spatially-disparate plant organs, such as roots and leaves
simultaneously, leading to variation in herbivore-induced BVOCs and consequences
for above- and belowground defences. Due to vascular connections, aboveground
herbivory can change the quantity and composition of root BVOCs and vice versa.
While the majority of studies focus on BVOC-mediated tritrophic interactions
above-ground, these relationships occur belowground as well and have important
effects on aboveground communities (Van der Putten et al. 2001). To our knowledge,
no studies to date have described the integration of above and belowground BVOC-
mediated tritrophic interactions in trees. While such interactions are likely to exist,
they will likely occur at different temporal time scales due to longer generation
times and contributing phenological factors. Recent studies focused on herbaceous
plants have shown that the vegetative portions of plants experiencing belowground
herbivory emit lower total BVOC emissions and produce different BVOC profiles
compared to plants solely attacked by an aboveground herbivore (Rasmann and
Turlings 2007). Thus, the presence of soil herbivory in this case appears to lower the
potential for defence, particularly when the belowground herbivore causes increased
damage as a function of development and size (Soler et al. 2007). Another study
demonstrated the effects of belowground herbivory on aboveground indirect defence
by showing that black mustard (Brassica nigra) plants experiencing root herbivory
emit high levels of sulfur-containing BVOCs, highly toxic for insects, and low levels
of (E)-“-farnesene, an attractant for parasitoids (Soler et al. 2007). In addition to
soil herbivores, mutualistic mycorrhizal associations can also effect aboveground
signalling, as in the case with parasitoids of aphids attracted to mycorrhizal plants
in the absence of their aphid hosts (Guerrieri et al. 2004). Because of signal cross-
talk, the timing of attack by above- and belowground herbivores can be crucial
when examining the extent to which communities are affected. Clearly, more studies
investigating BVOC responses to simultaneous attacks by above- and belowground
herbivores in forest species are needed.

2.3.4.3 Induced BVOCs Exhibit Diurnal Patterns

The release of herbivore-induced BVOCs occurs both locally and systemically

in space and also varies over time. For example, hybrid poplar leaves (Populus
trichocarpa  P. deltoides) attacked by forest tent caterpillars (Malacosoma
disstria) released similar characteristic blends of volatiles, including mono-,
sesqui-, and homoterpene compounds, that peaked during the light period (Arimura
et al. 2004). This diurnal pattern could be critical depending on parasitoid and
predator foraging patterns and the biologically-active compounds present. In
Nicotiana tabacum, several herbivore-induced BVOCs are exclusively released
at night and repel female moths (Heliothis virescens) searching for oviposition sites
(De Moraes et al. 2001). Because most parasitoids search during the day, these
2 BVOC-Mediated Plant-Herbivore Interactions 37

nighttime emissions may not be relevant as top-down defences, but the impact of
daytime indirect forcings and nighttime bottom-up effects may have significant
multiplicative consequences for herbivore densities. Currently, we lack studies
describing exclusive and additive diurnal ecological relationships mediated by
herbivore-induced emissions in tree systems.

2.3.4.4 Induced BVOCs: Immediate vs. Delayed Responses

A limitation of inducible volatile defences is the time-lag between damage,

induction, signalling, and the actual plant response (Dicke 2009). Upon feeding or
oviposition, the plant can emit BVOCs within seconds to minutes. Most of these
emissions, are not under control of the plant, but rather released as a consequence
of exposure to the atmosphere (Maffei et al. 2007). Compounds synthesized in
response to metabolic changes and involved in indirect plant defence are usually
expressed within hours or days following damage (Kunert et al. 2002). Thus,
parasitoids and predators must perceive and respond to these compounds within a
critical window of time before the benefits of effective host-location are missed.
Abiotic conditions and emissions from other organisms in the environment can
also influence herbivore-induced cues with important implications for top-down
controls over time, the scope of which is beyond this chapter. Rapid herbivore-
induced BVOCs active in plant resistance may stabilise insect densities; however,
delayed induced resistance, via foliar chemistry, potentially contributes to popula-
tion cycles (e.g., Roden and Mattson 2008). Some studies demonstrate that needles
of previously defoliated trees exhibit higher suitability for subsequent defoliator
generations (Lyytikäinen 1992; Clancy et al. 2004), while others have demonstrated
induced resistance after defoliation (Hódar et al. 2004; Šmits and Larsson 1999).
The susceptibility or resistance of previously defoliated trees depends on a number
of variables (i.e., tree age, intensity of defoliation, herbivore species, etc.), yet the
influence of BVOCs emitted during and after defoliation on higher trophic level
interactions has not been studied.

2.4 Insect Perception, Learning, and Evolutionary

Considerations

Insects must perceive and process enormous amounts of sensory information,

including chemical information, to locate their hosts within dynamic heterogeneous
environments (Vet 2001). BVOC infochemicals are sensed by olfactory sensory
neurons, primarily within antennae but also located within chemosensory sensilla
on other parts of the insect’s body, to aid in perceiving chemical signals in the
atmosphere. The ability to perceive BVOCs plays a key role in host location for
both herbivores and their natural enemies (Meiners et al. 2003), and many insects are
capable of identifying compounds present in the atmosphere at levels much lower
than some of our most sensitive analytical instruments.
38 A.M. Trowbridge and P.C. Stoy

The ability of an insect to perceive and respond to stimuli is not fixed, and can
change upon association with favorable or unfavorable stimuli. For example, female
parasitic wasps have a well-developed learning capacity to associate herbivore-
induced plant BVOCs with the presence of suitable hosts (de Boer and Dicke
2006). The ability of insects to exploit information from BVOCs can be both innate
(Gandolfi et al. 2003a; Steidle and van Loon 2003; Wang et al. 2003) and learned
(Dicke 1999; Wäckers and Lewis 1999). We briefly highlight studies that focus on
parasitoid learned behavior to emphasize the important role of behavioral ecology
in BVOC-mediated multitrophic interactions.
Regardless of the strict categorical descriptions, learning a given task in nature
likely involves a combination of stimulus-stimulus and stimulus-response asso-
ciations, allowing a parasitoid to take advantage of a variety of cues, including
unrewarding experiences, which might aid in future decisions and actions (Vet
et al. 2003). Learning behavior tends to be more prevalent in generalist parasitoids
(Geervliet et al. 1998) than in specialists (Mumm et al. 2005), suggesting that
specialist parasitoid species are more ‘hardwired’ when it comes to responding
to plant BVOCs expressed in response to their specific host. While this genetic
component may be beneficial in relatively stable environments, changes in climate
and/or community composition could confound host-associated signals with impor-
tant consequences for parasitoid adaptations and herbivore dynamics. Furthermore,
not all generalist species have the capability to learn (Tamò et al. 2006), which
complicates our ability to generalize parasitoid behavior in response to dynamic
chemical cues.
The ability of parasitoids to remember learned behavior also varies with time
and stimulus. Recollection of unrewarded activities often fade within a few hours to
days (Peri et al. 2006), but learning responses to odors of advantageous activities
tend to be more persistent (Takasu and Lewis 2003), and can even occur at
preimaginal stages before the adult stage (Gandolfi et al. 2003b). The coordination
of plant BVOC emissions with the window of parasitoid ‘memory’ is thus critical
for eliciting parasitoid response. Importantly, learning behavior has been found
to positively impact fitness (Dukas and Jun 2000) and contributes to coexistence
between parasitoids and their insect hosts (Hastings and Godfray 1999), which
emphasizes that learning plays an important role in not only chemical ecology, but
also in insect evolution and plant-insect coevolution.
BVOCs impact evolutionary pressures on herbivores and parasitoids through
their role in determining fitness. Biogenic BVOCs are involved in a range of
ecological functions (Fig. 2.1), and as a consequence, their role in plant evolution
is dynamic (Yuan et al. 2009). Adaptive explanations have been offered to address
the diversity of BVOCs found among and within plant families (Lerdau and Gray
2003; Wink 2003). It has also been argued that natural selection also exploits the
volatility of the compounds themselves and thereby the context in which they are
perceived by herbivores and their natural enemies (Peñuelas and Llusià 2004). The
precise ecological functions and evolutionary consequences of every BVOC are not
yet known (Niinemets et al. 2004), so their full contribution to plant-insect evolution
2 BVOC-Mediated Plant-Herbivore Interactions 39

has yet to be characterized. However, the importance of BVOCs to plant, herbivore,

and parasitoid fitness highlights their role in the evolution of each taxonomic group,
and their role in ecological signalling suggests that they play a substantial role in
coevolution among taxonomic groups.

2.5 Conclusions

BVOCs influence plant-insect interactions across multiple levels of ecological

organization and play active roles in bottom-up and top-down defences against her-
bivory. Important ecological consequences stem from feedbacks that exist between
constitutive and herbivore-induced BVOC emissions, herbivore and parasitoid
behavior, and the environment. Researchers are only beginning to uncover the role
BVOCs play in mediating tritrophic interactions and influencing coevolutionary
processes that exist between plants and insects. An improved understanding of the
impacts of global change on plant and insect ecology and evolution will help us
understand the full consequences of BVOC-mediated plant-insect interactions in
forested ecosystems, including the role of insects in recently observed forest die-
off (Raffa et al. 2008; Rhoades 1983). Models of surface-atmosphere exchange
have long had the capability to include BVOC dynamics (Guenther et al. 1995),
and their mechanistic representation of BVOC emissions is continuously being
improved (Monson et al. 2012). We suggest that including plant-insect interactions
into models of BVOC emissions will improve our understanding of the impacts of
these interactions on ecosystems, and to the entire Earth system.

Acknowledgments The authors acknowledge Russell K. Monson and Deane Bowers for en-
lightening conversations and sharing their enthusiasm for plant-insect interactions. PCS also
acknowledges funding from the National Science Foundation (‘Scaling ecosystem function: Novel
Approaches from MaxEnt and Multiresolution’, Division of Biological Infrastructure #1021095)
and the State of Montana.

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Chapter 3
The Biochemistry and Molecular Biology
of Volatile Messengers in Trees

Hamid Rajabi Memari, Leila Pazouki, and Ülo Niinemets

Abstract All tree species possess genes encoding terminal enzymes responsible
for volatile isoprenoid synthesis. However, only in some species, these genes are
expressed constitutively in leaves, while terpenoid emissions can be triggered by
abiotic and biotic stress factor in essentially all species. This chapter analyses
the biochemical diversity of volatile isoprenoid synthases and investigates the
genomic modifications responsible for constitutive volatile production in trees.
Plant terpenoids are up to three-domain proteins with either one active center in
monofunctional synthases, or two active centers in bifunctional synthases. There
is evidence of monophyletic origin of modern plant terpenoid synthases from
a three-domain synthase in an ancient progenitor followed by extensive gene
duplication and domain loss. The terpenoid synthase sequence similarity can be
low among distant plant groups, but terpenoid tertiary structure is remarkably
similar in different synthases, and this structural similarity is even conserved across
domains of life. However, only minor changes in active center structure can lead to
major changes in product profiles, indicating that presence of rich terpenoid genetic
diversity constitutes an important means for rapid evolutionary adaptations to novel
biotic interactions, and to new abiotic stresses in plant habitats.

H. Rajabi Memari
Genetic Engineering and Molecular Genetics, Biotechnology and Life Science Center and School
of Agriculture, Shahid Chamran University of Ahvaz, Golestan BLV, Ahvaz, Iran
L. Pazouki () • Ü. Niinemets
Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences,
Kreutzwaldi 1, Tartu 51014, Estonia
e-mail: [email protected]; [email protected]

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 47
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 3,
© Springer ScienceCBusiness Media Dordrecht 2013
48 H. Rajabi Memari et al.

3.1 Introduction

Humans have acknowledged the existence of terpenoids (isoprenoids) in plants

from ancient times without any information about their chemistry and role in
plants. The term terpene comes from the word turpentine, which is the resin
that oozes out from the bark of pine trees after wounding. This sticky resin is
rich in a variety of terpenoid compounds with widely differing chemical structure
and physico-chemical characteristics (Crozier et al. 2006). All terpenoids consist
of isoprene (C5) building blocks and are conditionally divided between primary
metabolites such as carotenoids and hormones and secondary metabolites such as
volatile terpenoids isoprene and monoterpenes (C10), and semivolatile terpenoids
sesquiterpenes (C15) and diterpenes (C20) fulfilling a variety of functions, some
of which are not yet fully understood (Modolo et al. 2009; Fineschi et al. 2013).
Altogether, these volatiles and semivolatiles constitute the most import class of
biogenic volatile organic compounds (BVOCs) (Kesselmeier and Staudt 1999;
Dudareva et al. 2004; Holopainen 2004; Guenther et al. 2006; Laothawornkitkul
et al. 2009).
Volatile terpenoids are released by different plant tissues including leaves, buds,
flowers, fruits and roots (Dudareva and Pichersky 2008), but vegetative plant parts,
in particular, leaves are believed to contribute the most to plant emissions due
to high leaf mass fraction and generally the highest emission rates per foliage
mass (Kesselmeier and Staudt 1999). Vegetative plant parts can release diverse
mixtures of terpenoids, including isoprene, monoterpenes, sesquiterpenes and some
diterpenes (Keeling and Bohlmann 2006a). These volatiles play different roles in
plants including enhancement of abiotic stress tolerance by serving as antioxidants
(Loreto et al. 2001b; Vickers et al. 2009a; Possell and Loreto 2013) and possibly also
by modifying membrane fluidity (Sharkey and Singsaas 1995; Singsaas et al. 1997;
Behnke et al. 2007), and serving as direct defences against omnivorous insects, e.g.,
oleoresin is a common direct defence in conifers against pathogens and herbivores
(Bohlmann and Croteau 1999; Raffa et al. 2005; Heijari et al. 2008; Chen et al.
2011). In addition, these volatiles serve as indirect defences participating in within-
plant and among-plant communication and stress priming and in communication
with herbivore enemies (Arimura et al. 2000; Dicke and Bruin 2001; Dudareva et al.
2006; Pieterse and Dicke 2007; Choudhary et al. 2008; Dicke et al. 2009).
Due to the importance of volatile terpenoids in plant life, there is a continuing
interest in plant terpenoid synthesis pathways and terpene synthase (Tps) genes as
targets to increase plant stress tolerance by enhanced terpenoid content and emission
of volatiles attracting herbivore parasites and predators (Pichersky and Gershenzon
2002; Degenhardt et al. 2003; Dudareva and Pichersky 2008). Understanding
structural, functional and evolutionary features of Tps family in trees can help to
discover defence systems in these plants and use this information for breeding and
tree protection in forestry. Furthermore, some of these volatiles or their derivatives
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 49

have cosmetic or medicinal properties, which have made them as important targets
for fragrance and pharmaceutical science and industry, for selection of promising
genotypes and genetic engineering to overexpress the pathway in the given or a new
host organism (Seigler 1998; Braun et al. 2001; Schepmann et al. 2001; Huang et al.
2004; Aharoni et al. 2005; Bohlmann and Keeling 2008; Saranitzky et al. 2009;
Trusheva et al. 2010). Also, in last decades, the pathways of volatiles have been
widely used and engineered to produce different types of biofuels (Peralta-Yahya
et al. 2012).
From air chemistry and climate perspective, volatile isoprenoids significantly
contribute to photochemical reactions in the atmosphere, participating in the forma-
tion of ozone, secondary organic aerosols and cloud condensation nuclei, thereby
altering air quality, and solar radiation penetration (Huff Hartz et al. 2005; Guenther
et al. 2006; Lee et al. 2006; Engelhart et al. 2008; Ashworth et al. 2013; Kulmala
et al. 2013). For example, the hemiterpene (C5) isoprene (2-methyl-1,3-butadiene)
is worldwide the most important volatile isoprenoid with global emissions about
440–660 Tg C year1 (Guenther et al. 2006; Ashworth et al. 2013). Isoprene
is emitted constitutively by leaves of several deciduous angiosperm tree species
like Salix spp., Populus spp., and Quercus spp., but also from several North-
American evergreen Quercus spp. (Kesselmeier and Staudt 1999). Furthermore,
some gymnosperms and a number of herb, moss and fern species release isoprene
as well (Sharkey and Yeh 2001; Sharkey et al. 2008). Carbon loss due to isoprene
emission in constitutive emitters is typically between 1 and 2 % of photosynthetic
carbon fixation, but this percentage may increase to more than 50 % under stress
conditions (Sharkey and Yeh 2001).
During the past decades, major progress has been made in identification and
functional characterization of volatile terpenoid biosynthesis genes, enzymes and in
metabolic engineering, and this has greatly contributed to improved understanding
of terpenoid biosynthesis (Crozier et al. 2006; Keeling and Bohlmann 2006b;
Bohlmann and Keeling 2008; Degenhardt et al. 2009; Nagegowda 2010; Chen et al.
2011). Latest developments in molecular techniques, such as new technologies for
identification of large genome parts up to full genomes and rapid assessment of plant
transcriptome are strongly contributing to identifying and isolating new terpenoid
genes, studying the synthase reaction mechanisms and understanding their function
in plants. In this chapter, we briefly describe the key pathways for immediate
substrates for plant terpenoid biosyntheses. Then we analyse isoprene, mono-
sesqui- and diterpene biosynthesis, characteristics of involved terpenoid synthases,
their regulation and corresponding gene families with special attention to trees
species. So far, the research on plant terpenoids has mainly focused on herbaceous
species. However, given that the bulk of volatiles released to the atmosphere is
believed to come from woody species, clearly there is a pressing need to gain more
insight into biochemical and genetic regulation of terpenoid biosynthesis in woody
species.
50 H. Rajabi Memari et al.

3.2 Terpenoid Biosynthesis

The huge chemical diversity of plant isoprenoids is formed by an extensive array of

enzymes that can be divided among three groups. The first group of enzymes serves
as the interface between primary and secondary metabolism, being responsible for
channeling of metabolites to terpenoid synthesis pathways (Modolo et al. 2009).
The second group of enzymes forms the terpenoid molecule scaffolds in terpenoid
pathways (Modolo et al. 2009). The third group of enzymes alters the terpenoid
backbones, resulting in new molecules with different biological activities (Modolo
et al. 2009), e.g., by hydroxylation, epoxidation, arylmigration, glycosylation,
methylation, sulfation, acylation, prenylation, oxidation, and reduction (Gowan
et al. 1995; Ro et al. 2005; Modolo et al. 2009). Here we briefly outline the
basic isoprenoid synthesis pathways. More detailed summary of terpenoid synthesis
pathways is provided by Rosenkranz and Schnitzler (2013) and Li and Sharkey
(2013b).

3.2.1 Main Biosynthesis Pathways: MVA and MEP/DOXP

All terpenoid compounds are synthesized from the same precursors: isopentenyl
diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) (Wanke
et al. 2001; Crozier et al. 2006; Bohlmann and Keeling 2008; Modolo et al.
2009). These precursors are synthesized by two different pathways. The cytosolic
mevalonic acid (MVA) pathway is present in most eukaryotes and is responsible for
the synthesis of C15 (sesquiterpenoids) and C30 (triterpenoids such as sterols) ter-
penoid compounds in plants (Gershenzon and Croteau 1993). The second recently
discovered 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate
pathway (MEP/DOXP pathway) is present in several prokaryotes and in plastids
of eukaryotic organisms (Rohmer et al. 1993; Eisenreich et al. 2004; Crozier et al.
2006). The MEP/DOXP pathway is responsible for the synthesis of isoprene (C5),
mono- (C10), di- (C20) and tetraterpenoids (C40) in plants (Lichtenthaler et al.
1997a, b; Modolo et al. 2009). The two pathways operate almost independently,
although there is a certain cross-talk among the two pathways at the level of IDP
(e.g., Hemmerlin et al. 2003; Laule et al. 2003).
Isoprenoid synthesis through MVA pathway starts with condensation of
three molecules of acetyl coenzyme A (acetyl-CoA) producing 3-hydroxy-3-
methylglutaryl-CoA (HMG-CoA). HMG-CoA is further reduced by HMG-CoA
reductase to mevalonic acid (MVA), which is phosphorylated by two kinases
forming mevalonate 5-diphosphate (MVADP). MVADP is converted into the
terpenoid precursor, IDP, by mevalonate diphosphate carboxylase (Gershenzon and
Croteau 1993; Eisenreich et al. 2004; Crozier et al. 2006). The cytosolic enzyme
isopentenyl diphosphate isomerase (IDI) further catalyzes the reversible conversion
between IDP and its isomer DMADP.
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 51

The plastidic MEP/DOXP pathway starts with the condensation of the substrates
pyruvate and glyceraldehyde 3-phosphate (GAP) to DOXP. Carbon skeleton
rearrangements and dehydration steps result in formation of 2-C-methyl-D-
erythritol 4-phosphate (MEP). MEP is further converted to 2-C-methyl-D-erythritol
2,4-cyclodiphosphate and to 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBDP).
Finally, HMBDP is converted both to IDP and DMADP by HMBDP reductase, and
the pool sizes of IDP and DMADP are further modulated by plastidic isopentenyl
diphosphate isomerase (Eisenreich et al. 2004; Crozier et al. 2006; Li and Sharkey
2013b).
In the past years, major progress has been made in characterizing the enzymes
of MEP/DOXP pathway in plants, but kinetic characteristics of all enzymes are
still not available, although the available evidence suggests that they resemble those
in bacterial counterparts (Harrison et al. 2013; Li and Sharkey 2013b). However,
differently from bacteria, MEP/DOXP pathway in plants can directly accept elec-
trons from photosynthetic electron transport chain. In particular, HMBDP synthase
and HMBDP reductase can accept electrons from ferredoxin (Seemann et al. 2006;
Seemann and Rohmer 2007), possibly explaining the tight coupling of MEP/DOXP
pathway to light reactions of photosynthesis in the chloroplasts. Overall, synthesis
of highly reduced terpenoids is energetically costly with synthesis of one isoprenoid
C5 residue needing fixation of 6 molecules of CO2 , and consuming 20 ATP, and 14
NADPH molecules (Sharkey et al. 2008), underscoring the need for high plastidic
ATP and NADPH status for DOXP/MEP pathway (Rasulov et al. 2011; Li and
Sharkey 2013a, b).

3.2.2 From Precursors to Terpenoids

DMADP is the substrate for the synthesis of the smallest isoprenoids, the hemiter-
penes isoprene and 2-methyl-3-buten-2-ol (MBO). IDP and DMADP are further
substrates for short-chain prenyltransferases (Bohlmann and Keeling 2008; Modolo
et al. 2009). The assembly of geranyl diphosphate, the backbone for monoterpenes,
by head-to-head addition of IDP and DMADP is catalyzed by GDP synthase.
Further farnesyl diphosphate (FDP), the backbone for sesquiterpenes, is formed by
condensing GDP and IDP by FDP synthase. Geranylgeranyl diphosphate (GGDP)
that is the substrate for diterpene synthesis is formed by condensing FDP and
IDP by GGDP synthase. Further, tri- and tetraterpenoids are made by head-to-
head condensation of two FDP and two GGDP molecules, respectively (Bohlmann
and Keeling 2008; Modolo et al. 2009). The resulting terpenoid polymers are used
as precursors by terpene synthases/cyclases and enter into synthesis of primary
terpenoid compounds such as sterols, phytyl-chain of chlorophyll and carotenoids
(Modolo et al. 2009). Terpenes can further be modified by hydroxylation and
oxidization by cytochrome P450-dependent enzymes (Ro et al. 2005; Keeling and
Bohlmann 2006a).
52 H. Rajabi Memari et al.

3.3 Terpenoid Synthases

Terpenoid synthases form a diverse class of enzymes catalyzing formation of

molecules with different chain length, including hemiterpene synthases (C5),
monoterpene synthases (C10), sesquiterpene synthases (C15), and diterpene syn-
thases (C20). Being often the end-points of the pathway, they are the key terminal
flux-controlling enzymes. So far, over 60,000 members of the terpenoid family
are recognized, and a broad grouping of structures has been clarified (http://
dnp.chemnetbase.com/) (Xie et al. 2012). There has been a major progress in
understanding the function and structure of terpenoid synthases catalyzed by
rapid developments in molecular biology techniques allowing for dissection of
the structure of terpenoid synthase gene families and heterologous expression and
study of recombinant terpenoid synthases (Degenhardt et al. 2009). A number of
model terpene synthases has been characterized in detail (Hyatt et al. 2007; Köksal
et al. 2010, 2011a, b; McAndrew et al. 2011; Zhou et al. 2012), but we are just
starting to understand the size and structure of terpene synthase families in key
organisms (Sect. 3.4). Furthermore, there is less information on tree terpenoid
synthases than on synthases in herbaceous species, except perhaps for gymnosperms
(Degenhardt et al. 2009). Nevertheless, the progress has been amazingly rapid as
new biotechnology techniques are providing strong tools to understand the complex
biochemical function and regulation of genes involved in the terpenoid pathways.
The emergence of new high throughput techniques such as deep sequencing together
with developments in computational bioinformatics is allowing shedding light on
previously hidden aspects of genomes, transcriptomes, proteomes, metabolomes
and finally terpenomes with unprecedented detail (Christianson 2008; Cane and
Ishida 2012). Here we introduce the basic contemporary methods to study terpenoid
synthases and analyse the basic functional structure of terpenoid synthases with
emphasis on tree enzymes.

3.3.1 Identification and Analysis of the Functional Activity

of Terpenoid Synthases

Due to simultaneous expression of multiple terpenoid synthases in plant tissues and

relatively low product specificity of most synthases (Sect. 3.3.3.4), functional anal-
ysis of terpenoid synthases based on enzyme purification from crude leaf extracts
is difficult, although a lot of pioneering work has been conducted using partially
purified enzymes extracted from plants (e.g., Croteau and Karp 1977; Croteau et al.
1978; Dehal and Croteau 1988). Development of molecular biology techniques for
identification and isolation of individual terpenoid synthases has opened completely
new vistas for studying biochemistry, structure, genetics and evolution of terpenoid
synthases. Development of RNA sequencing platforms in recent years using deep-
sequencing technologies have changed the transcriptomics world and sometimes
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 53

predicate the death of micro-array and other transcriptome analysis technologies like
serial analysis of gene expression (SAGE, Velculescu et al. 1995), cap analysis gene
expression (CAGE, Shiraki et al. 2003), and massively parallel signature sequencing
(MPSS, Brenner et al. 2000). However, the high throughput technologies have some
disadvantages like high cost, inability to detect transcripts for isoforms and splice
variation (Wang et al. 2009; Myllykangas et al. 2012).
The workflow for functional characterization of given terpene synthase typically
consists of determination of the sequence of terpenoid synthase genes either on
the basis of mRNA or genomic DNA, heterologous expression in a host system
of the sequenced gene and functional characterization of the recombinant protein.
Ultimately, the function can be further studied in a transgenic plant model system.
Here these basic steps are briefly outlined.

3.3.1.1 Identification of Terpenoid Synthase Genes

In the infancy of terpenoid molecular and functional studies, identification of

terpene synthase genes was a highly tedious task due to lack of information on
homologous sequences for degenerate primer construction. Now, as more and more
synthases have been sequenced, the rich existing genetic information allows for
more rapid progress, albeit identification of terpenoid synthases with low level
of homogeneity with those described so far, especially in organisms with little
genome coverage is still difficult (Cane and Ishida 2012). By now, 51 genomes of
higher plants (38 published) have been fully sequenced (as of Sept. 4, 2012, http://
genomevolution.org/wiki/index.php/Sequenced plant genomes), making it possible
to identifiy putative terpenoid synthases by genome mining. Genome sequence
analysis of Arabidopsis thaliana has identified about 30 terpene synthase genes in
this model organism (Aubourg et al. 2002), but in several vascular plant species
much larger terpene synthase families have been detected (Sect. 3.4.2, Martin
et al. 2010; Li et al. 2012), especially by using the widest range of terpenoid
synthase sequences possible in homology searches (Li et al. 2012). In particular,
information about the size and structure of tree terpene synthase gene families
has been exponentially increasing since the first tree, Populus trichocarpa (Tuskan
et al. 2006), genome completion, followed by other tree genome projects including
Carica papaya (Ming et al. 2008), Malus domestica (Velasco et al. 2010), Phoenix
dactylifera (Al-Dous et al. 2011), and Pyrus bretschneideri (Wu et al. 2013). New
bioinformatics tools such as the use of profile-based hidden Markov models rather
than pairwise searchers makes it possible to identify terpene synthase genes carrying
remote sequence homology, thereby identifying putative terpene synthases with
potential structural similarity of basic conserved functional domains (Gough et al.
2001; Wilson et al. 2009; Cane and Ishida 2012).
Although the number of fully sequenced genomes is rapidly increasing, the
genome of only 11 tree species has been sequenced, most of them tree crops.
Among sequenced trees, only Populus trichocarpa and Eucalyptus grandis can be
conditionally considered as ‘wild plants’. Thus, techniques alternative to genome
54 H. Rajabi Memari et al.

mining need to be applied to most tree species. New high throughput techniques
(e.g., next generation sequencing, Liu et al. 2012) have opened up possibilities
for fast characterization of transcriptome, and identification of expressed terpenoid
synthases by transcriptome mining. Conifers are characterized by a particularly
rich blend of terpene volatiles, suggesting a highly diverse terpenoid family, but
conifer genome is especially complex, and full sequences of first conifer genomes
are unlikely in the near future, although a number of genome projects has started
(see e.g., http://pinegenome.org/). Thus, relatively few terpenoid synthases have
been functionally characterized in conifer species so far, although more than in
angiosperm trees (e.g., Wildung and Croteau 1996; Bohlmann et al. 1997, 1999;
Hall et al. 2011). In these pioneering studies, different techniques were used to
identify conifer terpene synthases, such as cDNA library screening and similarity-
based PCR (Bohlmann et al. 1998a). Expressed sequence tag (EST) libraries
have been available lately for loblolly pine (Pinus taeda) (Allona et al. 1998),
Japanese cedar (Cryptomeria japonica) (Ujino-Ihara et al. 2000), white spruce
(Picea glauca), interior spruce (P. glauca  P. engelmannii), and Sitka spruce
(Picea sitchensis) (Holliday et al. 2008). Moreover, lots of attempts are currently
in progress, trying to identify and characterize more terpene synthases in conifers
using a combination of targeted cDNA cloning, large amounts of ESTs and full-
length cDNA mining (Byun McKay et al. 2003; Miller et al. 2005; Keeling et al.
2011) as well as using other techniques such as bacterial artificial chromosome
(BAC) technique (Hamberger et al. 2009). Use of these new methods has allowed
identification of large terpenoid synthase families in several tree species, e.g., 69
actively expressed Tps genes (including monoterpene, sesquiterpene and diterpene
synthases) have been identified in Picea species (Keeling et al. 2011).

3.3.1.2 Heterologous Expression of Tree Terpenoid Synthases

in E. coli and in Plants

Functional characterization of terpene synthases is usually carried out by het-

erologous expression in Escherichia coli (Table 3.1 for a selected list of tree
terpenoid synthases expressed in E. coli). However, expression of terpene synthase
genes in E. coli carries potential problems. Some terpene synthase proteins are
produced in cytosol, but are targeted to chloroplastic compartment, therefore having
chloroplastic signal peptides. These transient sequences should be removed before
cloning and expression in the host for sufficiently high expression of recombinant
protein (Phillips et al. 2003). On the other hand, codon usage of eukaryotic genes
is different from prokaryotic host, and the eucaryotic genome also contains rare
codons (Fig. 3.1). The comparison between codon usages in the angiosperm Salix
discolor and Escherichia coli shows the mean difference close to 30 % in codon
usage, whereas the difference between the angiosperm Populus trichocarpa and the
gymnosperm Abies grandis is about 10 %. Thus, for high level of expression, co-
transformation of a plasmid encoding the rare tRNAs (e.g., for Arg) is needed (Hohn
1999; Martin et al. 2004). The codon optimization for gene expression of tree Tps
Table 3.1 List of selected tree terpenoid synthases expressed and characterized in Escherichia coli host system
UniProtKB/
Gene Swiss-Prot
Terpenoid synthase familya entry Species Expression host Substrate Terpenoid productsb References
Hemiterpenes (C5 )
Isoprene synthase Tps-b Q50L36 Populus alba E. coli origami DMADP Isoprene (100 %) Sasaki et al.
B (DE3) (2005)
Isoprene synthase Tps-b Q9AR86 Populus x E. coli TG1 DMADP Isoprene (100 %) Miller et al. (2001)
canescens
2-Methyl-3-buten-2-ol Tps-d1 F5CJS6 Pinus E. coli BL21 DMADP Methylbutenol (99 %), Gray et al. (2011)
(MBO) synthase sabiniana (DE3)-RIL isoprene (1 %)
Monoterpenes (C10 )
()-Camphene Tps-d1 Q948Z0 Abies E. coli GDP ()-camphene (54 %), Bohlmann et al.
synthase grandis BL21(DE3) ()-’-pinene (32 %), (1999)
()-limonene
(C)-Carene synthase 1 Tps-d1 F1CKI6 Picea E. coli GDP (C)-3-carene (42 %), Hall et al. (2011)
sitchensis ’-terpinolene (27 %),
(C)-sabinene (11 %),
()-“-phellandrene,
myrcene, ’-terpinene,
”-terpinene, ()-’-pinene,
’-thujene
(C)-Carene synthase 2 Tps-d1 F1CKI8 Picea E. coli GDP (C)-3-carene (64 %), Hall et al. (2011)
sitchensis ’-terpinolene (15 %),
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees

(C)-sabinene (7 %),
()-“-phellandrene,
’-terpinene, ”-terpinene,
myrcene
()-Limonene Tps-d1 O22340 Abies E. coli GDP ()-limonene (>90 %), Bohlmann et al.
synthase grandis BL21(DE3) ()-’-pinene, ()-“-pinene (1997)
(continued)
55
 56

Table 3.1 (continued)

UniProtKB/
Gene Swiss-Prot
Terpenoid synthase familya entry Species Expression host Substrate Terpenoid productsb References
()-Limonene/()-’- Tps-d1 Q9M7C9 Abies E. coli BL21(DE3) GDP ()-limonene (35 %), Bohlmann et al.
pinene synthase grandis ()-’-pinene (24 %), (1999)
()-“-phellandrene (20 %),
()-“-pinene (11 %),
()-sabinene (10 %)
(C)-Limonene synthase Tps-b Q8L5K1 Citrus limon E. coli BL21- GDP (C)-limonene (99 %) Lücker et al.
CodonPlus-RIL (2002)
(C)-Limonene synthase Tps-b Q8L5K3 Citrus limon E. coli BL21- GDP (C)-limonene (99 %) Lücker et al.
CodonPlus-RIL (2002)
Limonene synthase Tps-d1 Q675L1 Picea abies E. coli BL21- GDP ()-limonene (88 %), myrcene, Martin et al.
CodonPlus ()-’-pinene, (C)-limonene (2004)
Linalool synthase Tps-d1 Q675L2 Picea abies E. coli BL21- GDP ()-linalool (97 %), Martin et al.
CodonPlus (C)-linalool, E-“-ocimene (2004)
Myrcene synthase Tps-d1 O24474 Abies E. coli XL1-Blue GDP Myrcene (100 %) Bohlmann et al.
grandis (1997)
Myrcene synthase Tps-b Q93X23 Quercus ilex E. coli TG1 GDP Myrcene (97 %), limonene, Fischbach et al.
“-pinene (2001)
Myrcene synthase Tps-d1 Q675K9 Picea abies E. coli BL21- GDP Myrcene (100 %) Martin et al.
CodonPlus (2004)
()-“-Phellandrene Tps-d1 Q9M7D1 Abies E. coli BL21(DE3) GDP ()-“-phellandrene (52 %), Bohlmann et al.
synthase grandis ()-“-pinene (34 %), (1999)
()-’-pinene (9 %),
()-limonene
(C)-’-Pinene synthase Tps-d1 Q84KL3 Pinus taeda E. coli BL21(DE3) GDP (C)-’-pinene (97 %), Phillips et al.
()-’-pinene (2003)
H. Rajabi Memari et al.
()-’-Pinene synthase Tps-d1 Q84KL6 Pinus taeda E. coli GDP ()-’-pinene (79 %), Phillips et al.
BL21(DE3) ()-“-pinene, (2003)
()-limonene,
(C)-limonene,
(C)-camphene,
()-camphene,
(C)-’-pinene,
(C)-“-pinene
()-“-Pinene synthase Tps-b Q8L5K2 Citrus limon E. coli BL21- GDP ()-“-pinene (81 %), Lücker et al.
CodonPlus- sabinene (11 %), (2002)
RIL ()-’-pinene,
()-limonene
()’/“-Pinene Tps-d1 Q675L3 Picea abies E. coli GDP ()-’-pinene (57 %), Martin et al.
synthase BL21(DE3) ()-“-pinene (27 %), (2004)
“-phellandrene (11 %),
myrcene
Pinene synthase Tps-d1 Q6XDB5 Picea E. coli BL21- GDP ()-’-pinene (65 %) Byun McKay et al.
sitchensis CodonPlus ()-“-pinene (18 %), (2003)
(DE3)
()-Pinene synthase Tps-d1 O24475 Abies E. coli XL1- GDP ()-’-pinene (42 %), Bohlmann et al.
grandis Blue/E. coli ()-“-pinene (58 %) (1997)
XLOLR
(C)-Sabinene Tps-d1 F1CKJ1 Picea E. coli GDP (C)-sabinene (54 %), Hall et al. (2011)
synthase sitchensis ’-terpinolene (31 %),
()-’-pinene,
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees

’-terpinene,
()-“-phellandrene,
”-terpinene, myrcene,
(C)-3-carene
(continued)
57
Table 3.1 (continued)
58

UniProtKB/
Swiss-Prot
Terpenoid synthase Gene familya entry Species Expression host Substrate Terpenoid productsb References
”-Terpinene synthase Tps-b Q8L5K4 Citrus limon E. coli BL21- GDP ”-terpinene (71 %), Lücker et al.
CodonPlus- ()-limonene (9 %), (2002)
RIL ()-’-pinene,
(C)-“-pinene,
terpinolene, ’-thujene,
(C)-limonene,
’-terpinene
Terpinolene synthase Tps-d1 Q9M7D0 Abies E. coli GDP Terpinolene (42 %), Bohlmann et al.
grandis BL21(DE3) ()-’-pinene (18 %), (1999)
()-limonene (11 %),
()-“-pinene (10 %)
()-’-Terpineol Tps-d1 Q84KL4 Pinus taeda E. coli GDP ’-terpineol (57 %), Phillips et al.
synthase BL21(DE3) limonene (28 %), (2003)
terpinolene (8 %),
“-pinene, ’-pinene,
myrcenec
(C)-’-Terpineol Tps-b B5A434 Santalum E. coli C41 GDP (C)-’-terpineol (44 %), Jones et al. (2008)
synthase album ()-limonene (33.6 %),
E-geraniol, linalool,
myrcene, ()-’-pinene,
(C)-sabinene,
’-terpinolene
Sesquiterpenes (C15 )
’-Bisabolene synthase Tps-d3 O81086 Abies E. coli FDP E-’-bisabolene (>99 %) Bohlmann et al.
grandis XL1-Blue (1998a)
GDP (C)-limonene (main
product)
E-’-Bisabolene Tps-d3 Q675L6 Picea abies E. coli BL21- FDP E-’-bisabolene (100 %) Martin et al.
H. Rajabi Memari et al.

synthase CodonPlus (2004)

’-Farnesene synthase Tps-b Q84LB2d Malus E. coli DH5’ FDP E,E-’-farnesene (>99 %), Pechous and
domestica Z,E-’-farnesene (<1 %) Whitaker
(2004)
GDP E-“-ocimene (68 %), Pechous and
(Z)-“-ocimene (32 %) Whitaker
(2004)
’-Farnesene synthase Tps-b Q84LB2e Malus E. coli BL21- FDP E,E-’-farnesene (96 %), Green et al. (2007)
domestica CodonPlus- Z,E-’-Farnesene
RIL (<1 %)
GDP E-“-ocimene (90 %), Green et al. (2007)
linalool, myrcene
E,E-’-Farnesene Tps-d1 Q675K8 Picea abies E. coli BL21- FDP E,E-’-farnesene (100 %) Martin et al.
synthase CodonPlus (2004)
()-Germacrene D Tps-a Q64K29 Populus E. coli BL-21- FDP ()-germacrene D (79 %), Arimura et al.
synthase trichocarpa CodonPlus “-caryophyllene, (2004)
x P. deltoides ’-humulene,
alloaromadendrene and
4 non-identified
terpenoids
”-Humulene synthase Tps-d2 O64405 Abies E. coli FDP ”-humulene (dominant), Steele et al. (1998)
grandis XL1-Blue sibirene, longifolene,
“-himachalene,
”-himachalene,
’-himachalene
Longifolene synthase Tps-d2 Q675L0 Picea abies E. coli BL21- FDP Longifolene (61 %), Martin et al.
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees

CodonPlus ’-longipinene (15 %), (2004)

longicyclene,
E-“-farnesene,
longiborneol,
cyclosativene,
“-longipinene
(continued)
59
 60

Table 3.1 (continued)

UniProtKB/
Swiss-Prot
Terpenoid synthase Gene familya entry Species Expression host Substrate Terpenoid productsb References
Diterpenes (C20 )
Abietadiene synthase Tps-d3 Q38710 Abies E. coli BLR/E. coli GGDP Abietadiene (mixture of Peters and Croteau
grandis XL1-Blue double bond isomers) (2002) and Stofer
Vogel et al. (1996)
Isopimaradiene Tps-d3 Q675L5 Picea abies E. coli BL21- GGDP Isopimara-7,15-diene Martin et al. (2004)
synthase CodonPlus (100 %)
Levopimaradiene Tps-d3 Q947C4 Ginkgo E. coli GGDP Levopimaradiene (main Schepmann et al.
synthase biloba product) (2001)
Levopimaradiene Tps-d3 Q675L4 Picea abies E. coli BL21- GGDP Levopimaradiene (37 %), Martin et al. (2004)
synthase CodonPlus abietadiene (32 %),
neoabietadiene (23 %),
palustradiene
Taxadiene synthase Q41594 Taxus E. coli JM109/ GGDP Taxadiene (mixture of Wildung and Croteau
brevifolia E. coli double bond isomers) (1996), Williams
BL21(DE3) et al. (2000) and
Jin et al. (2005)
a
According to Bohlmann et al. (1998b), Byun McKay et al. (2003), and Martin et al. 2004
b
Only minor products 1 % are demonstrated
c
Dominated by () isomer, except for ’-pinene (100 % (C)-’-pinene)
d
GenBank accession number AY182241.2, expressed with C-terminal Myc-tag (Pechous and Whitaker 2004)
e
GenBank accession number AY787633, expressed with N-terminal His-tag (Green et al. 2007)
H. Rajabi Memari et al.
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 61

genes can also be achieved by using suitable host strains. Rosetta host strains are
BL21 derivatives designed to enhance the expression of eukaryotic proteins that
contain rare codons (like tree codons) which are seldom used in E. coli. This strategy
can facilitate overexpression and characterization of different tree Tps genes in
E. coli (Kane 1995).
The heterologous expression of Tps synthase in E. coli is followed by disruption
of cellular contents of E. coli cultures carrying the transgenic construct, enzyme
purification whenever needed, and assay of enzymatic activity (Martin et al. 2004;
Sasaki et al. 2005; Majdi et al. 2011). Typically, the functional characterization
of the protein involves incubation of the soluble recombinant enzyme with the
substrate GDP, FDP, or GGDP in the presence of Mg2C and/or Mn2C , and analysis
of the product profiles by gas chromatography – mass spectrometry (GC-MS) and
identification of terpenoids by authentic standards (Bohlmann et al. 1997; Martin
et al. 2004; Falara et al. 2011). Typically, an overlay of pentane or other hydrophobic
solvent is used to trap the hydrophobic reaction products formed in the aqueous
reaction mixture (e.g., Martin et al. 2004), or volatiles can also be sampled in the
head-space using a solid-phase micro extraction (SPME) fiber (e.g., Falara et al.
2011) or using sample air from the headspace with a GC preconcentration trap (e.g.,
Fischbach et al. 2001).
Although heterologous expression offers an unique opportunity to work with
the isolated protein without other potentially interfering proteins, the kinetic
characteristics of the recombinant enzyme can differ from the native enzyme, but
not necessarily substantially, e.g., as demonstrated by similar Km values for GGDP
for the diterpene taxadiene native and recombinant enzyme (Williams et al. 2000).
Also, enzyme characteristics can depend on the transgenic construct, e.g., farnesene
synthase expressed with C-terminal Myc-tag (Pechous and Whitaker 2004) and
N-terminal His-tag (Green et al. 2007) had somewhat different product profiles
(Table 3.1). Expression of poplar isoprene synthase either with N-terminal or
C-terminal His-tag resulted in altered pH and temperature dependence and substrate
specificity (Schnitzler et al. 2005). Thus, some caution is warranted when making
inferences on the performance of native enzyme on the basis of measurements with
recombinant protein.
Heterologous expression in E. coli can be followed by characterization of the
functional role of the protein in plants as discussed in detail by Rosenkranz and
Schnitzler (2013). For example, transgenic Arabidopsis model systems expressing
isoprene synthase from white poplar (Populus alba) (Sasaki et al. 2007), grey
poplar (P. x canescens) (Loivamäki et al. 2007a, 2008) and kudzu (Pueraria lobata)
(Velikova et al. 2011), and transgenic tobacco (Nicotiana tabacum) expressing
P. alba isoprene synthase gene (Vickers et al. 2009b, 2011) are available. Also,
herbaceous model systems overexpressing certain mono-, sesqui- and diterpenes
have been constructed (El Tamer et al. 2003; Besumbes et al. 2004; Wu et al.
2006). However, no such model systems were yet available for trees. In this book,
Rosenkranz and Schnitzler (2013) first time describe successful introduction of
poplar isoprene synthase gene into silver birch (Betula pendula), providing a new
exciting model to test the role of isoprene synthase in plants.
62 H. Rajabi Memari et al.
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 63

3.3.2 Conserved Motifs and Functional Domains

of Terpenoid Synthases

3.3.2.1 Conserved Motifs

According to the mechanism of catalysis, terpenoid synthases can be separated

among two major classes. In the case of class I enzymes, the catalysis is initiated
by metal-triggered ionization of the substrate diphosphate group, while for class
II enzymes, the catalysis is initiated by protonation of an epoxide ring or carbon–
carbon double bond (Christianson 2006, 2008; Aaron and Christianson 2010; Cao
et al. 2010). In both cases, a highly reactive carbocation is formed that enters
into isomerization and cyclization steps until the catalysis is terminated by either
proton elimination or nucleophilic capture from the final carbocation (Aaron and
Christianson 2010; Cao et al. 2010; Köksal et al. 2011a). Thus, the primary
difference among the class I and class II terpenoid synthases is the initial step of
the catalysis. These differences in catalytic mechanism also reflect different origin
of terpenoid synthases with all type I synthases sharing the ‘type I synthase fold’, an
’-helical structure, containing a core of bundled anti-parallel ’-helices, while type
II enzymes have a characteristic “’-barrel” structure (Aaron and Christianson 2010;
Cao et al. 2010).
These differences in the catalytic mechanism are reflected in differences in con-
served motifs and functional domains. Class I terpenoid synthases are characterized
by the aspartate (D)-rich DDXXD and (N/D)DXX(S/T)XXXE (N is aspargine,
S is serine and T is threonine, X can be any amino acid) motifs (Fig. 3.2) that
are responsible for binding of divalent metal ions, in particular Mg2C or Mn2C ;
these metal ions are responsible for diphosphate elimination from the substrate,
resulting in carbocation formation (Starks et al. 1997; Degenhardt et al. 2009;
McAndrew et al. 2011). The DDXXD motif is typically located at the entrance
position of the catalytic site and plays a prominent role in positioning the substrate
for catalytic reaction, while (N/D)DXX(S/T)XXXE motif is located at the opposite
site of the active site entry, with the underlined amino acids coordinating the metal
cations (Little and Croteau 2002; Degenhardt et al. 2009; Cao et al. 2010; Köksal
et al. 2011a). Mutational analysis of these motifs in diterpene abietadiene synthase

J
Fig. 3.1 Comparison of codon usage between (upper panels) the bacterium E. coli (red bars) and
the angiosperm Salix discolor (black bars) and between (lower panels) the gymnosperm Abies
grandis (red bars) and the angiosperm Populus trichocarpa (black bars). Relative adaptiveness is
an index of usage of synonymous codons that scales the frequency of use of given codon relative to
the optimal codon (most frequently used codon with the greatest translation efficiency) (Sharp and
Li 1987). This index is calculated for a given codon j and for given amino acid i, wij , as xij /ximax ,
where xij is the frequency of the use of the given codon and xi,max is that for the optimal codon
(Sharp and Li 1987). wij facilitates comparison of the codon usage in different proteins and among
different organisms. Codon frequency is based on NCBI GenBank (www.kazusa.or.jp/codon/). The
data was extracted and analysed by graphical codon usage analyzer (http://gcua.schoedl.de/)
64 H. Rajabi Memari et al.

Fig. 3.2 Two DDXXD metal binding motifs in sesquiterpene synthases in the gymnosperm grand
fir (Abies grandis). The first sequence is for •-selinene synthase (ag4, UniProtKB/Swiss-Prot
entry O64404), the second for ’-bisabolene synthase (ag1, O81086) and the third for ”-humulene
synthase (ag5, O64405) (The sequences were aligned using UniProt protein sequence database,
http://www.uniprot.org/align/)

in Abies grandis demonstrated that mutations in metal-coordinating amino acids

strongly reduced the enzyme catalytic activity (Zhou and Peters 2009). However,
for some terpene synthases, e.g., the sesquiterpene synthases ”-humulene synthase
(ag5) and •-selinene synthase (ag4) from Abies grandis, the sequence information
indicates that (N/D)DXX(S/T)XXXE motif is replaced by another DDXXD motif
(Back and Chappell 1996; Steele et al. 1998).
Class II terpene synthases contain a conserved DXDD motif which is responsible
for protonation of the double bond in the initial reaction step (Cao et al. 2010;
Köksal et al. 2011a; Zhou et al. 2012). Mutations in the central aspartic acid
render the active site completely non-functional (Christianson 2006; McAndrew
et al. 2011). As the diphosphate cleaving activity is missing in class II terpenoid
cyclases, they commonly yield diphosphorylated products such as the diterpenoid
copalyl diphosphate (CDP) formed by CDP synthase (Keeling et al. 2010; Köksal
et al. 2011a) unless they use non-diphosphorylated substrates such as the bacterial
squalene-hopene synthase (Wendt et al. 1997). Exceptionally, class I monoterpene
bornyl diphosphate synthase from Salvia officinalis forms a diphosphorylated
product, but in this unusual reaction mechanism, diphosphate residue is first cleaved
and then again reincorporated (Whittington et al. 2002).

3.3.2.2 Functional Domains of Terpenoid Synthases

Terpenoid synthases consist of up to three functional domains, ’-, “ and ”-domain.

’-domain bears class I terpene synthase activity, “-domain class II terpene synthase
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 65

Fig. 3.3 Structure of selected tree terpenoid synthases: (a) hemiterpene isoprene synthase from
Populus x canescens (Protein Data Bank, http://www.rcsb.org/pdb, PDB ID: 3N0F) (Köksal
et al. 2010), (b) sesquiterpene •-cadinene synthase from Gossypium arboreum (PDB ID: 3G4F,
Gennadios et al. 2009), (c) sesquiterpene ’-bisabolene synthase from Abies grandis (PDB ID:
3SAE, McAndrew et al. 2011), and (d) diterpene abietadiene synthase from Abies grandis (PDB
ID: 3S9V, Zhou et al. 2012). Different colours correspond to ’- (green, C-terminus), “- (red,
N-terminus) and ”-domain (blue). The active site for the terpenoid synthases in (a–c) is in the
’-domain. The bifunctional abietadiene synthase has two active sites, one in the ’-domain and the
other in the “-domain. The illustrations were generated by Protein Homology/analogY Recognition
Engine 2.0 (PHYRE 2.0, http://www.sbg.bio.ic.ac.uk/phyre2) (Kelley and Sternberg 2009) that
uses the protein structure library stored in Protein Data Bank (http://www.rcsb.org)

activity, while ”-domain does not have any known catalytic site (Aaron and
Christianson 2010; Cao et al. 2010; Köksal et al. 2011a). Although the domains
form clearly separate folds in protein tertiary structure (Fig. 3.3), in the protein
amino acid sequences, ”-domain sequence is typically embedded within the “-
domain sequence (Wendt et al. 1997; Zhou et al. 2012). In nature, ’-domain
synthases and “-”-domain synthases can be found in several organisms such as
bacteria and fungi. For example, sesquiterpene pentalenene synthase from the
actinobacterium Streptomyces (Cane et al. 1994; Lesburg et al. 1997; Caruthers et al.
2000), sesquiterpene aristolochene synthase from the fungus Penicillium roqueforti
(Caruthers et al. 2000) and sesquiterpene trichodiene synthase from the fungus
Fusarium sporotrichioides (Rynkiewicz et al. 2001) are ’-domain only class I ter-
penoid synthases, whereas triterpenoid squalene-hopene cyclase from the firmicute
Alicyclobacillus acidocaldarius (Wendt et al. 1997; Siedenburg and Jendrossek
2011) and diterpene tuberculosinol diphosphate synthase from the actinobacterium
Mycobacterium tuberculosis (Nakano and Hoshino 2009) are “-”-domain terpenoid
synthases. To our knowledge, none of the plant species possesses “-”-domain
synthases, and most of the plant terpene synthases either contain ’-“-domains or
all the three domains, ’-“-” (Cao et al. 2010; Hillwig et al. 2011). These plant
terpenoid synthases have been postulated to originate from a fusion of an ’-domain
66 H. Rajabi Memari et al.

type and a “-”-domain type terpenoid synthase in an ancient progenitor (Morrone

et al. 2009; Cao et al. 2010, Sect. 3.4.1), resulting in formation of ’-“-” domain
synthases followed by ”-domain loss, yielding ’-“ domain synthases (Hillwig et al.
2011).
Until recently, it was thought that plants do not possess single, ’-domain,
synthases. However, it was just discovered that phylogenetically old spikemoss
Selaginella muellendorffii has both “plant-type” ’-“-”- and ’-“-type terpenoid
synthases, and “microbial-type” ’-domain only terpenoid synthases, fundamentally
altering our understanding of the structure of plant terpene synthase families
(Li et al. 2012).
All proteins can be classified based on the functional domains. Structural Classi-
fication of Proteins (SCOP, http://scop.mrc-lmb.cam.ac.uk/scop/) database provides
an hierarchical way to systematize proteins among classes, folds, superfamilies
and families (Murzin et al. 2001; Andreeva et al. 2008). A terpenoid synthase
superfamily embraces all terpenoid synthase protein domains sharing a common
evolutionary origin (Wilson et al. 2009). As ’-domain and “- or “-”-domains of
terpenoid synthases have different evolutionary origin (Morrone et al. 2009; Cao
et al. 2010, Sect. 3.4.1), plant terpenoid synthase protein domains are divided
between two superfamilies, the superfamily Terpenoid synthases that includes
the ’-domains of terpenoid synthases (for most plant terpenoid synthases the
relevant domain family is ‘Terpenoid cyclase C-terminal domain’) and superfamily
Terpenoid cyclases/protein prenyltransferases that includes “- or “-”-domains of
terpenoid synthases (family: ‘Terpenoid cyclase N-terminal domain’). As plant
terpenoids commonly consist of either ’-“- or ’-“-”-domains, they typically belong
simultaneously to both superfamilies. ”-domain, sequence of which is embedded
within the “-domain sequence, is classified together with “-domain in SCOP (Gough
et al. 2001), The superfamily can be highly diverse at the level of amino acid
sequences, but the structures of terpenoid synthases within given superfamily are
broadly similar (Fig. 3.4, Sect. 3.3.2.3).

3.3.2.3 Structural Alignment of Terpenoid Synthases in Trees

The three-dimensional protein structure reveals ultimate level of structural infor-

mation directly related to its function. It is possible that any two proteins with
low amino acid sequence similarity still have close structural homology, suggesting
similar functional activity. In fact, for terpenoid synthases this appears to be the case.
The terpenoid synthases are present in multiple domains of life, and the sequence
homology can be quite low even within the same domain, and in particular, across
the domains of life (Bohlmann et al. 1998b; Chen et al. 2011; Cane and Ishida
2012; Li et al. 2012). As noted in Sect. 3.3.1.1, genomes can be screened for
proteins sharing even remote homology with powerful bioinformatics computational
algorithms, identifying new terpenoid synthases (see also Sect. 3.3.2.2). This way,
the spikemoss “microbial-type” terpenoid gene family has been recently discovered
(Li et al. 2012).
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 67

Fig. 3.4 Pairwise structural alignment of tree terpenoid synthases (a–c) and alignment of proteins
structures across different domains of life (d, e). The proteins aligned in (a–c) are as described in
Fig. 3.3. The sesquiterpene pentalenene synthase in (d) is from the actinobacterium Streptomyces
sp. UC5319 (PDB ID: 1PS1, Lesburg et al. 1997), the diterpene taxadiene synthase in (e) is from
the gymnosperm Taxus brevifolia (PDB ID: 3P5R, Köksal et al. 2011b), and the triterpenoid
squalene-hopene cyclase is from the firmicute Alicyclobacillus acidocaldarius (PDB ID: 1SQC,
Wendt et al. 1997). The alignment was conducted with the Protein Data Bank alignment tool (http://
www.rcsb.org) (Berman et al. 2000; Prlic et al. 2010). Pentalenene synthase has only ’-domain,
isoprene and •-cadinene synthases have ’-“-domains, squalene-hopene cyclase has “-”-domains
and ’-bisabolene, abietadiene and taxadiene synthases have ’-“-”-domains. The proteins are
oriented as in Fig. 3.3 with the ’-domain at the top and “- or ”-domain at the bottom. The olive
colour is for the first aligned synthase and cyan for the second, and the grey parts stand for non-
aligned sequence components. In (c) the sequence similarity is 52, and 97 % of •-cadinene synthase
(sequence length 515 amino acids, AA) is structurally aligned with isoprene synthase (531 AA).
In (b), the sequence similarity is (36 %) and 95 % of isoprene synthase is aligned with abietadiene
synthase (755 AA). In (c), the sequence similarity is 48 and 100 % of abietadiene synthase is
aligned with ’-bisabolene synthase (780 AA). In (d), the sequence similarity is 22, and 92 % of
pentalenene synthase (304 AA) is aligned with isoprene synthase. In (e), the sequence similarity is
17 and 67 % of squalene-hopene synthase is aligned with taxadiene synthase (750 AA)

At present, only a few X-ray crystal structures of plant Tps proteins are available
(Starks et al. 1997; Kampranis et al. 2007; Degenhardt et al. 2009; Gennadios
et al. 2009; Köksal et al. 2011a, b). In the case of trees, crystal structures are
available for isoprene synthase from grey poplar (P. x canescens) (Köksal et al.
2010), ’-“-domain sesquiterpene synthase •-cadinene synthase from tree cotton
(Gossypium arboreum) (Gennadios et al. 2009) and ’-“-”-domain sesquiterpene
’-bisabolene synthase from Abies grandis (McAndrew et al. 2011), and two ’-“-”-
domain diterpene synthases, bifunctional (class I and class II) abietadiene synthase
from A. grandis (Zhou et al. 2012) and monofunctional (class I) taxadiene synthase
from Taxus brevifolia (Köksal et al. 2011b) (Fig. 3.3). The three gymnosperm
synthases are all from terpene synthase family Tps-d1, while poplar isoprene
synthase belongs to Tps-b family and tree cotton •-cadinene synthase to Tps-
a family (Li and Sharkey 2013b for discussion of classification of terpenes into
gene families). For ’-“-domain monoterpene synthases, the crystal structures are
available only for herbs, including Tps-b family monoterpene limonene (Hyatt et al.
2007) and bornyl diphosphate (Whittington et al. 2002) synthases.
68 H. Rajabi Memari et al.

Despite limited coverage, available crystal structures have provided major insight
into the structure of catalytically active sites, metal binding motifs, substrate
recognition and subsequently the function of proteins. Data on protein three-
dimensional structure can be employed for protein structural alignment. Structural
alignment is a robust tool to compare proteins’ tertiary structure and reveal hidden
evolutionary relationships, especially for proteins with high evolutionary distance,
and consequently with little similarity in their nucleic acid or amino acid sequences.
In such cases, the evolutionary relationships between proteins cannot be easily
detected by standard sequence alignment techniques, but structural alignment can
be used to gain insight into functional relationships among proteins with low
sequence homology. For example, despite low level of sequence similarity, poplar
isoprene synthase (Tps-b family) and tree cotton •-cadinene synthase (Tps-a family)
exhibit high structural similarity (Fig. 3.4a). High structural similarity is also evident
among poplar isoprene synthase and grand fir abietadiene synthase (Tps-d family)
’-“-domains (Fig. 3.4b). Furthermore, significant structural similarity is evident
between plant and microbial terpenoid synthases. Albeit the sequence similarity
is only about 20 % (Fig. 3.4d, e), ’-domain alignment of poplar isoprene synthase
and bacterial pentalenene synthase, and “-”-domain alignment of Pacific yew (Taxus
brevifolia) taxadiene synthase and bacterial squalene-hopene cyclase are remarkably
good. Overall, this evidence again emphasizes the strong structural similarity within
given domains of terpene synthases across plants and even across the kingdoms of
life (Aaron and Christianson 2010; Cao et al. 2010; Cane and Ishida 2012).

3.3.3 Characteristics of Key Plant Terpenoid Synthases

3.3.3.1 Isoprene Synthase

Isoprene synthase (IspS) is the terminal enzyme completing chloroplastic isoprene

synthesis through MEP/DOXP pathway (Sharkey and Yeh 2001; Sharkey et al.
2008). IspS crystal structure for recombinant protein from grey poplar (P. x
canescens) was recently characterized, and it was demonstrated that it is a classic
two domain, ’-“, terpenoid synthase. The C-terminal class I terpenoid synthase fold
(’-domain) possesses the catalytic activity, while the N-terminal class II terpenoid
synthase domain (“-domain) possesses no known catalytic activity (Köksal et al.
2010). Formation of isoprene from DMADP occurs through a syn-periplanar
elimination mechanism via an allylic carbocation intermediate as in other class I
terpenoid synthases (Köksal et al. 2010). The enzyme requires Mg2C for catalytic
activity and has a relatively broad alkaline pH optimum between 7 and 8.5, and a
temperature optimum between 40 and 45 ı C (Monson et al. 1992; Sasaki et al. 2005;
Schnitzler et al. 2005). IspS has a high Km value for DMADP in vivo of ca. 0.3 mM
(Rasulov et al. 2009), and there is evidence of allosteric regulation (Schnitzler et al.
2005) and competitive inhibition by GDP, the substrate for monoterpenoid synthases
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 69

(Köksal et al. 2010). Multiple isoprene synthase genes have been demonstrated in
some poplar species, but the role of these paralogous genes is not yet clear (Vickers
et al. 2010). Further details of isoprene synthase are provided in Rosenkranz and
Schnitzler (2013) and Li and Sharkey (2013b).
All of the sequenced IspS synthase genes so far have suggested to posses specific
chloroplastic signal sequences (Miller et al. 2001; Sasaki et al. 2005; Sharkey
et al. 2005; Fortunati et al. 2008; Vickers et al. 2009a, 2010). Localization of
IspS to chloroplasts in constitutive emitters has also been confirmed by chloroplast
extractions (Wildermuth and Fall 1996; Wildermuth and Fall 1998), immunogold-
labelling (Schnitzler et al. 2005), and chloroplast-allocation of green fluorescent
protein fused with isoprene synthase in transformed constructs (Sasaki et al. 2005).
In fact, transgenic tobacco (Nicotiana tabacum) expressing isoprene synthase in
cytosol appeared to be essentially void of isoprene emission (Vickers et al. 2011).

3.3.3.2 Terpene Synthases

Terpenes are synthesized by terpene synthases from one of three common prenyl
diphosphate precursors formed by the fusion of DMADP with one or more isopen-
tenyl diphosphate (IDP) molecules, catalyzed by prenyltransferases (Chappell 1995;
Koyama and Ogura 1999; Dewick 2002).
Typically, tree mono- and sesquiterpene synthases are ’-“-domain proteins with
only the ’-domain (class I) terpene synthase active site functional (Aaron and
Christianson 2010; Cao et al. 2010). These terpene synthases are ca. 550–650
amino acids long with sesquiterpene synthases functionally active in the cytosol
being characteristically 50–70 amino acids shorter than hemi- and monoterpene
synthases that possess a N-terminal plastid-targeting sequence (Bohlmann et al.
1998a; Degenhardt et al. 2009).
Most diterpene synthases and some sesquiterpene synthases are three-domain
proteins, 800–870 amino acids long. Again, diterpene synthases are longer than
three-domain sesquiterpene synthases due to N-terminal plastid-targeting sequence
(Bohlmann et al. 1998a), but some of these three-domain sesquiterpene synthases
might contain N-terminal signal sequence (Bohlmann et al. 1998b; Martin et al.
2004). Despite having three domains, only class I terpene synthase active site in
the ’-domain is functional in sesquiterpenes due to lack of DXDD motif as in
’-bisabolene synthase in Abies grandis (McAndrew et al. 2011). In the case of
diterpenes, commonly either only class I or class II terpene synthase activity is
present and the other active site is rendered non-functional (Keeling et al. 2010).
Among the diterpenes, taxadiene synthase in Pacific yew (Taxus brevifolia) has
class I terpene synthase activity (Köksal et al. 2011b), while ent-copalyl diphosphate
(CDP) synthase in Arabidopsis has class II terpene synthase activity (Köksal et al.
2011a).
A few bifunctional diterpene synthases having both class I and class II terpene
synthase activities have been reported to date in trees, including abietadiene
70 H. Rajabi Memari et al.

synthase from Abies grandis (Vogel et al. 1996; Peters et al. 2001; Zhou et al. 2012),
cis-abienol synthase from Abies balsamea (Zerbe et al. 2012b), levopimaradiene
synthases from Gingko biloba (Schepmann et al. 2001), and Picea abies (Martin
et al. 2004) and abietadiene/levopimaradiene synthase from Pinus taeda (Ro and
Bohlmann 2006). In bifunctional diterpene synthases, “-domain with class II
activity typically forms a diphosphorylated diterpenoid intermediate (e.g., CDP)
that freely diffuses to the second, class I active site in the ’-domain, where the
diphosphate group is cleaved and given diterpenoid (or typically, a spectrum of
terpenoids) is formed (Zhou et al. 2012).
While it is generally thought that mono- and diterpene synthases are functional
in plastids, and sesquiterpene synthases in cytosol, there is surprisingly little
information on subcellular location of terpene synthases other than the presence
or absence of plastid targeting sequences in the expressed proteins (Nagegowda
2010). Available evidence does suggest that monoterpenes are functionally active
in plastids (Nagegowda 2010), but ’-terpineol synthase in Magnolia appeared to
be targeted both to chloroplasts and mitochondria (Lee and Chappell 2008), and
there are terpene synthases, capable of formation of both mono- and sesquiterpenes
depending on substrate (Sect. 3.3.3.3), that can be localized in the cytosol or in
the plastids (Aharoni et al. 2004; Nagegowda et al. 2008). On the other hand,
while sesquiterpene synthases are generally located in the cytosol, presence of
N-terminal residue in some three-domain sesquiterpene synthases in conifers Abies
grandis and Picea abies (Bohlmann et al. 1998b; Martin et al. 2004) suggests
that they might be targeted to chloroplasts. Recently, three-domain sesquiterpene
santalene/bergamotene synthase from wild tomato (Solanum habrochaites) was
shown to be targeted to chloroplasts, suggesting that sesquiterpene synthesis can
occur via MEP/DOXP pathway (Sallaud et al. 2009). On the other hand, further
biochemical modifications in plastid-synthesized terpenoids, e.g., by cytochrome
P450-dependent oxidases typically take place in cytosol (Haudenschild et al. 2000;
Ro and Bohlmann 2006; Hamberger et al. 2009). Cleary, more work is needed
to gain insight into subcellular location of various terpene synthases, but the
evidence suggests that three-domain sesquiterpene synthases with strong homology
to diterpenoid synthases could be located in plastids.
In general, terpene synthases have much higher substrate affinity than isoprene
synthase with Km values as low as 0.9 M (linalool synthase, Pichersky et al. 1995),
7.6 M (linalool/nerolidol synthases, Nagegowda et al. 2008) to 84 M (myrcene
synthase, Fischbach et al. 2001) for GDP and 1.4 M (santalene synthase, Jones
et al. 2011) – 23 M (linalool/nerolidol synthases, Nagegowda et al. 2008) for
FDP, and 3–10 M for GGDP (taxadiene synthase, Hezari et al. 1995; Williams
et al. 2000). Typically, the pH optimum of terpene synthases is neutral to somewhat
alkaline between 6 and 7.5, but the optimum tends to be sharper than that for
isoprene synthase (Cori and Rojas 1985; Alonso and Croteau 1993; Bohlmann
et al. 1998b). The temperature optimum of terpene synthases seems to be lower
than that for isoprene synthase with reported values around 40 ı C for conifer Picea
abies and broad-leaved evergreen angiosperm Quercus ilex (Fischbach et al. 2000,
2001).
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 71

3.3.3.3 Substrate Specificity of Terpenoid Synthases

Typically, monoterpene synthases use geranyl diphosphate (GDP), sesquiterpene

synthases farnesyl diphosphate (FDP) and diterpene synthases geranylgeranyl
diphosphate (GGDP) as the only substrate (e.g., Fischbach et al. 2001; Martin
et al. 2004 for the tests of multiple substrates). However, a few plant terpene
synthases have reported to form terpenoids of different chain length depending
on substrate. Among these multifunctional enzymes, ’-bisabolene synthase from
the gymnosperm tree Abies grandis forms sesquiterpene E-’-bisabolene with FDP
as substrate and monoterpene (C)-limonene with GDP (Bohlmann et al. 1998a,
Table 3.1). ’-Farnesene synthase from the angiosperm tree Malus domestica forms
sesquiterpene E,E-’-farnesene with FDP and E-“-ocimene with GDP (Pechous and
Whitaker 2004; Green et al. 2007, Table 3.1). For both sesquiterpene synthases,
the enzymes preferably form sesquiterpenes when FDP and GDP are supplied
simultaneously (Pechous and Whitaker 2004; Green et al. 2007). Sesquiterpene
santalene synthase from Santalum species can form a spectrum of monoterpenes
when incubated with GDP, but the affinity to GDP is much less than to FDP (Jones
et al. 2011). In snapdragon (Antirrhinum majus) (Nagegowda et al. 2008) and straw-
berry (Fragaria ananassa) (Aharoni et al. 2004) nerolidol/linalool synthases were
shown to form monoterpenes linalool and other acyclic monoterpenes with GDP
and nerolidol with FDP. Recently a myrcene/isoprene synthase was characterized
in Humulus lupulus that formed myrcene with GDP, and isoprene with DMADP
(Sharkey et al. 2013).
Capacity to form different products depending on substrate has also been
reported for bacterial terpenoid synthases and seems to be widespread in nature
(Hamano et al. 2002; Siedenburg and Jendrossek 2011). So far, the available
evidence suggests that this trait is rare among plants, but clearly it is recommended
to routinely use multiple substrates to test for broad-spectrum substrate use capacity
in functional characterization of terpene synthases.

3.3.3.4 Product Specificity of Terpenoid Synthases

Plants produce a huge variety of terpenoids with diverse composition among and
within species. This high variety reflects expression of several terpene synthases
(Sects. 3.3.3.2 and 3.4.2) as well as multiple products of single terpene synthases.
For example, ”-humulene synthase from Abies grandis has been shown to produce
52 sesquiterpene olefins (Steele et al. 1998). However, all synthases have a certain
specificity to form some main products with greater probability. Typically, the syn-
thases also are stereospecific, forming preferably certain stereoisomers (Table 3.1,
Croteau 1987; Prosser et al. 2004; Christianson 2006, 2008; Degenhardt et al.
2009). In fact, the capacity to produce multiple products is not universal, and some
terpenoid synthases are almost completely specific, forming only one product or
nearly so (Table 3.1). For example, isoprene synthases tend to form only isoprene,
with the only known exception being a myrcene/isoprene synthase that can form
72 H. Rajabi Memari et al.

acyclic monoterpenes when GDP is the substrate (Sect. 3.3.3.3, Sharkey et al. 2013),
but there are also several highly specific mono-, sesqui- and diterpene synthases
(Table 3.1).
Predicting product profiles based on the terpenoid synthase full amino acid
sequences is currently not possible as the correlative patterns are weak (Degenhardt
et al. 2009). Even with the genes having high sequence homology, some enzymes
might produce multiple products while others form one single product; highly
homologous enzymes forming multiple products might produce strongly different
product spectra (Degenhardt et al. 2009). Depending on the reaction mechanism
(Markovnikov vs. anti-Markovnikov addition to a double bond), more or less stable
carbocation can be formed in the initial reaction steps, thereby affecting the potential
range of products formed (Christianson 2006). Also, active site structure, presence
of certain fixed and protected dipoles that can stabilise the carbocation, and steric
limitations play an important role in the product formation (Christianson 2006,
2008). Clearly, more X-ray crystal structures of tree terpene synthases are needed
to gain further insight into the reaction mechanisms in the highly diverse terpenoid
synthase family in trees.

3.4 Origin and Size of Terpenoid Synthase Gene Families

The vast array of different terpenoid compounds in nature results both from the
low product specificity of many terpenoid synthases (Sect. 3.3.3.4) and from large
number of terpenoid synthases present in most plant species studied. Presence
of a vast number of terpenoid synthase genes can reflect divergent evolutionary
paths for catalytic activities of terpenoid synthase genes leading to variations
in terpenoid compounds formed (Bohlmann et al. 1998a; Aubourg et al. 2002;
Dornelas and Mazzafera 2007), and resulting in novel functions that increase
species resistance to insects, pathogens, and herbivores (Trapp and Croteau 2001)
as well as to abiotic stresses (Owen and Peñuelas 2005; Fineschi et al. 2013;
Possell and Loreto 2013), overall increasing species fitness. On the other hand,
synthesis of similar compounds by phylogenetically widely distant plant species,
e.g., among angiosperms and gymnosperms possessing terpenoid synthases with
low homogeneity, also indicates convergent evolution of terpenoid synthases, and
in some cases, multiple events of evolution and loss of capacity to form the
given compound such as isoprene (Bohlmann et al. 1998a; Aubourg et al. 2002;
Dornelas and Mazzafera 2007; Monson et al. 2013). Here we analyse the evidence
of the origin of terpenoid synthases in plants and the size of terpenoid synthase
gene families with emphasis on tree terpenoid synthases. Evolution of terpenoid
synthases, including classification of plant terpenoid synthase gene families, is
addressed in other chapters of the book (Fineschi et al. 2013; Li and Sharkey
2013b).
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 73

3.4.1 Origin of Plant Terpenoid Synthase Gene Families

It has been suggested that all modern plant terpenoid synthases originate from a
common three-domain, ’-“-”-diterpene synthase in an ancient progenitor, followed
by independent gene duplications and evolution of new genes in different organ-
isms. As such an ancestor gene in plants, a bifunctional ent-copalyl diphosphate
synthase/ent-kaurene synthase (PpCPS/KS) of the moss Physcomitrella patens has
been suggested (Hayashi et al. 2006). This enzyme produces ent-kaurene which is
a common precursor of gibberellins and endogenous diterpenes derived from this
compound (Hayashi et al. 2010). The active site in the “-domain has class II terpene
synthase activity and is responsible for ent-copalyl diphosphate synthesis from
GGDP, while the active site in the ’-domain forms ent-kaurene from ent-copalyl
diphosphate. Additional bifunctional kaurene synthases have been discovered in the
phylogenetically old species liverwort Jungermannia subulata (Kawaide et al. 2011)
and spikemoss Selaginella muellendorffii (Li et al. 2012).
In phylogenetically younger gymnosperms and angiosperms, ent-kaurene is pro-
duced by two distinct mono-functional enzymes, ent-copalyl diphosphate synthase
(CPS) and ent-kaurene synthase (KS) (Keeling et al. 2010; Zerbe et al. 2012a).
Monofunctional diterpene synthases still have ’-“-” three-domain structure, but the
active site in either ’- or “-domain has lost the functional activity. As noted in
Sect. 3.3.3.2, such three-domain structure is characteristic also to some sesquiter-
pene synthases, but most sesquiterpene, and all mono- and hemiterpene synthases
have lost ”-domain sequence and have ’-“ domain configuration. Phylogenetic
analyses suggest that the formation of ’-“-domain terpenoid synthases via loss
of the ”-domain has occurred several times during evolution (Hillwig et al.
2011).
An interesting question is what are the immediate ancestors for the ’-domain
and “-”-domain in the common higher plant terpenoid ’-“-” ancestor? Analysis of
the phylogenetic signals separately for ’- and “-domains across a broad-spectrum
of plant terpenoid synthases actually does not confirm the postulated origin of all
terpenoid synthases from the moss Physcomitrella patens diterpene synthase gene
(Fig. 3.5). If this were the ancestor of all multidomain plant terpenoid synthases,
phylogenetic signals in ’- and “-domains should reflect this, but the sequence
homologies rather suggest that there must have been another common ancestor
for moss, liverwort, spikemoss, gymnosperm and angiosperm terpene synthases
(Fig. 3.5). Interestingly, ent-copalyl diphosphate and ent-kaurene synthases have
been discovered in the bacterium Bradyrhizobium japonicum (Morrone et al. 2009).
These terpenoid synthases share some homology with plant terpenoid synthases,
and it has been suggested that there might be even an ancient progenitor for modern
terpenoid synthases across the domains of life (Morrone et al. 2009).
Particularly interesting is the position of recently discovered spikemoss
Selaginella muellendorffii ’-domain only ‘microbial’-type terpene synthases
(Li et al. 2012). These sequences carry homology with both ‘modern plant’
74 H. Rajabi Memari et al.
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 75

multi-domain terpenoid synthases and microbial ’-domain only synthases, possibly

providing the missing link to modern plant terpenoid origin. As more information
of genomes becomes available, we will be possibly able to more clearly identify the
origin of terpenoid synthases in plants.

3.4.2 Size of Terpenoid Synthase Gene Families

Given the possible monophyletic origin of ‘higher plant type terpene synthases’,
continuous and extensive gene duplication followed by functional and structural
specialization is responsible for the high diversity of terpenoid synthase gene
families in plants (Fryxell 1996; Bohlmann and Keeling 2008; Chang and Duda
2012). The Viridiplantae have the largest terpenoid synthase families among living
organisms (Fig. 3.6), but the size of terpenoid gene families varies strongly among
plant species. According to genome mining by profile-based hidden Markov models,
the size of terpenoid gene family varies from one in the moss Physcomitrella patens
to 86 in the angiosperm tree Eucalyptus grandis (Fig. 3.6). These estimates based
on similarity screens of full genomes broadly agree with independent estimates
in other studies (Bohlmann and Keeling 2008; Chen et al. 2011). However, it is
difficult to detect genes with low level of homogeneity. For instance, in Selaginella
muellendorffii, 16–18 terpene synthases have been suggested based on genome
screens for higher plant, ’-“- or ’-“-”-domain, terpene synthases (Chen et al. 2011),

J
Fig. 3.5 Phylogenetic trees of selected tree terpene synthases (red font corresponding to gym-
nosperms and green font to angiosperms) and representative plant (blue font, moss Physcomitrella
patens and spikemoss Selaginella muellendorffii) and bacterial and fungal outgroups based
on ’-domain (C-terminal part of the sequence, (a) and “-domain (N-terminal part of the
sequence, (b). It has been suggested that all modern plant terpenoids originate from a fusion of
’-domain and “-”-domain terpenoid synthases in an ancient progenitor (Morrone et al. 2009;
Cao et al. 2010). Thus, ’-domain and “-domain might carry somewhat different phylogenetic
signals (Sharkey et al. 2013). UniProtKB/Swiss-Prot entry codes are also given with species name
(http://www.uniprot.org/). Isoprene and MBO (2-methyl-3-buten-2-ol) are hemiterpenes; linalool,
limonene, myrcene, “-pinene and ’-terpineol are monoterpenes; aristolochene, ’-bisabolene,
caryophyllene, “-farnesene, germacrene D, humulene, longifolene, T-muurolol, and pentalenene
are sesquiterpenes; abietadiene, levopimaradiene, and ent-kaurene are diterpenes; and hopene and
squalene are triterpenes. The microbial synthases in (a) only have ’-domain as the S. muellendorffii
‘microbial type’ terpene synthase D8LD3 (Li et al. 2012), while the microbial and fungal
squalene/hopene synthases in (b) have “-”-domains only. The domains were separated using
Abies grandis three-domain abietadiene synthase domain structure (Q38710, Zhou et al. 2012)
as a seed. After alignment, signal peptides were deleted, and whenever pertinent, the sequence
part corresponding to ”-domain was deleted. ’-domain and “-domain parts of the sequence were
separately aligned, truncated after alignment to a common length, and the trees were constructed by
MEGA5 software using the maximum likelihood method (Tamura et al. 2011). The numbers next
to the branches refer to the actual bootstrap values of branches and characterize the reliability of
the branching (bootstrap consensus trees are demonstrated). The higher the score, the more reliable
is the branching at that point (Tamura et al. 2011)
76 H. Rajabi Memari et al.

32 32 29 29
33 29

Mimulus guttatus

Capsella rubella
Solan

lor
33 29

Or

bico
yza
34

Th
28

um ly
eo

hum
Ar

sa
ab

br

s
tiva aca
34 ido 27

ay
om

copes

Sorg
ps

m
a

a
is ic
tal

a
c
th

Ze
37 ali i 27
ia

icum
Malu an tar us

o
s do a S e s ativ
mes is
39 tica cum 27
Cu a rapa
Brassic
39 Fragaria vesca 26
a 24 Medicago trunc
46 Aquilegia caerule atula 22
Viridiplantae
s
47 nsi arpa 20
s ine c
trus ho
Ci tric
sum
ondii

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a

Eu
48 19
Physcomitrella
tin

Eucalyptus grandis

pu Pr
tre

Po
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ma
tub

56 17
Cari

us
sc

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um

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67 11
71 8
86 1

Fig. 3.6 Protein superfamily ‘Terpenoid synthases’ domains in Viridiplantae clade analysed using
SUPERFAMILY 1.75 (http://supfam.cs.bris.ac.uk/SUPERFAMILY/) that includes essentially all
protein domains from all available sequenced organisms, including 36 sequenced Viridiplantae
genomes (Gough et al. 2001; Wilson et al. 2009). Superfamily is defined as a collection of domains
(functional units of proteins) having structural and functional evidence of common evolutionary
ancestor. Plant terpenoid synthases characteristically have either two (’ and “) or three (’, “ and ”)
domains (Figs. 3.3 and 3.4, Hillwig et al. 2011; Köksal et al. 2011a, b). The homologous domains
in SUPERFAMILY are identified by hidden Markov models, which is a profile-based method
with high selectivity (Gough et al. 2001). Here the protein domains from the domain family of
‘Terpenoid cyclase C-terminal domain’ that contains the ’-domain of the plant ’-“- and ’-“-”-
domain terpenoid synthases are demonstrated. In the figure, the radius of the circle reflects the size
of the terpenoid synthase superfamily in logarithmic scale (numbers inside denote the number of
terpenoid synthase domains in the given organism), the inner circle shows the average number of
terpenoid synthase domains in Viridiplantae. Trees are denoted by grey filling

but recently 48 putative “bacterial” type, ’-domain only synthases were identified
in Selaginella genome, and functional activity of several of them was characterized
(Li et al. 2012). Thus, we might have vastly underestimated the size of terpene gene
families in plants.
The largest terpenoid synthase gene families among sequenced Viridiplantae
are observed in trees (Fig. 3.6). Among top seven sequenced plants with the
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 77

greatest terpenoid synthase families, four, Eucalyptus grandis, Citrus clementina,

C. sinensis and Populus trichocarpa are trees, and Vitis vinifera is a woody vine
(Fig. 3.6, Martin et al. 2010). Furthermore, in addition to raw data generated by
genome projects, functional characterization and analysis of terpenoid genes by
transcriptome mining has highlighted large terpenoid gene families also in several
gymnosperms (Martin and Bohlmann 2005; Chen et al. 2011; Keeling et al. 2011).
However, some tree species such as Carica papaya have small terpenoid synthase
gene families (Fig. 3.6). Currently, the functional implications of variations in
the size of terpenoid synthase gene families have not yet been fully elucidated,
especially in trees. However, it is reasonable to expect that presence of multiple
terpenoid synthase genes forming different compounds and compound spectra and
presence of paralogs with similar compound spectra, but potentially with differing
regulatory promoter elements is associated with greater diversity of volatile product
profiles and more diverse response patterns to biotic and abiotic stresses (Keeling
et al. 2008; Bohlmann et al. 2011). Given that only minor modifications, at the
level of single amino acid or a few amino acids, may be needed to change the
terpenoid synthase function (Kampranis et al. 2007; Keeling et al. 2008; Hall et al.
2011), presence of duplicated genes can constitute an important gene pool for
rapid adaptation to new biotic interactions, ‘new’ abiotic stresses in given plant
habitat, and novel stress combinations or to changes in stress severities, thereby
helping plants to adapt to their environment (Hall et al. 2011). In addition to
variations among species, there is evidence of important within-species variation in
the terpenoid gene family, in agreement with adaptive genomic modifications (Hall
et al. 2011; Gonzales-Vigil et al. 2012).

3.5 Regulation of Terpenoid Synthesis

The quantities of metabolites ultimately produced are influenced by genetic features

and environmental signals. So, the metabolite contents in plants are dynamic and
controlled by internal and external stimuli. These changes are regulated by gene
expression networks involved in targeting pathways to specific cells and organs and
determining the overall expression level of the pathway (Grotewold 2008). Studying
one or some genes is not enough for having a deep insight into such networks across
a variety of tissues and treatments. Transcriptome and metabolome data provided
by next generation technologies have opened new opportunities for understanding
the biosynthesis and regulation of plant metabolites (Crispin and Wurtele 2013).
Perhaps with this comprehensive data, we will be able to start unresolving the
complex regulatory networks, not only focusing on the synthesis of terpenoid end-
products, but addressing the entire cascade of events altering profoundly plant
‘normal’ metabolism, from stress signals to elicitation of pathways.
Substrate-level regulation of terpenoid synthesis has been addressed in the
chapters of Li and Sharkey (2013b) and Monson (2013). Here we analyse the
regulation of terpenoid synthesis driven by variations in gene expression. Terpenoid
78 H. Rajabi Memari et al.

synthase genes show a high variety of temporal and spatial expression patterns;
some of these compounds are constitutively expressed (Steele et al. 1998), some
of them induced by both biotic and abiotic stresses (Yin et al. 1997; Byun-McKay
et al. 2006; Tholl 2006; Fares et al. 2008; Chen et al. 2011), some are expressed
in leaf mesophyll and leaf surface trichomes (Köllner et al. 2004; van Schie et al.
2007; Falara et al. 2011), and others in flowers (Chen et al. 2003; Dudareva et al.
2003; Falara et al. 2011). This level of control has been termed as ‘genetic control’
(Monson 2013).

3.5.1 ‘Constitutively’ Expressed Synthases

Regulation of terpenoid synthase gene expression is controlled by the regulatory

elements in the gene promoter region that interact with a variety of transcription
factors. Rosenkranz and Schnitzler (2013) provide an in-depth overview of the
poplar isoprene synthase promoter region (PcIspS). Isoprene synthase in emitting
species is a constitutively expressed gene. However, PcIspS contains circadian, heat
and stress-dependent regulatory elements (Loivamäki et al. 2007b; Cinege et al.
2009) and thus, expression of isoprene synthase varies during the day, during the
season and is responsive to changes in temperature (Mayrhofer et al. 2005; Sasaki
et al. 2005; Wiberley et al. 2008, 2009). So far, it is however, unclear how isoprene
synthase activity is regulated in plants grown under different atmospheric CO2
concentrations (Wilkinson et al. 2009; Sun et al. 2012).
There is much less information available on regulation of expression of terpene
synthases. In particular, there are several constitutively expressed terpene synthase
genes analogous to isoprene synthase, such as myrcene synthase in broad-leaved
evergreen Mediterranean oaks (Fischbach et al. 2001), which seem to be regulated
similarly to isoprene synthase, i.e., responding to ambient temperature and light
conditions (Fischbach et al. 2002; Staudt et al. 2003), and might be responsive to
CO2 as well (Loreto et al. 2001a), but so far the promoter regions of these enzymes
have not been characterized.
In most conifers, there are also several constitutively expressed enzymes for
oleoresin formation. Oleoresin in these species is accumulated in specific resin
ducts or galls in the bark, sapwood, and needles, and serves as a major physico-
chemical defence barrier against insects and pathogens (Bohlmann and Croteau
1999; Trapp and Croteau 2001; Martin and Bohlmann 2005; Raffa et al. 2005).
Release of oleoresin after insect attack repels and deters insects; emissions of mono-
and sesquiterpenes provide indirect defence against herbivores, but diterpenes and
terpene oxidation products provide direct defence by forming a physical barrier
at the place of insect attack (Martin et al. 2003; Miller et al. 2005; Raffa et al.
2005). The promoter regions of constitutively expressed terpenoid synthases in
conifers have not yet been characterized. In herbs, there has been some progress
in identifying promoters targeting the gene expression to glandular trichomes
(Tissier 2012), but again, terpenoid-specific promoters have not yet been identified.
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 79

Information about terpenoid-specific transcription factors is currently also very

limited (Tholl 2006; Chen et al. 2011), and obviously understanding terpenoid
synthase regulation should constitute a priority for future studies.

3.5.2 Stress-Induced Terpene Synthases

Emissions of a large number of mono- and sesquiterpenes are elicited in response

to numerous biotic stresses such as herbivory, oviposition and fungal inoculation,
but also to abiotic stresses through activation of signalling responses triggered
by reactive oxygen species. Stress-induced production of traumatic resin plays a
prominent role as a physical and toxic direct defence to insects and pathogens
(Martin et al. 2002; Miller et al. 2005; Raffa et al. 2005), while volatile terpenoid
emissions can serve as repellents to herbivores and attractants to enemies of
herbivores (Thaler 2002; Hilker et al. 2005; Miller et al. 2005; Raffa et al. 2005;
Dicke et al. 2009). There is a plethora of examples of stress-elicited modifications
in terpenoid synthesis. For detailed consideration of these responses, we refer to the
chapters of Trowbridge and Stoy (2013) and Holopainen et al. (2013) and the review
of Keeling and Bohlmann (2006b) and only briefly mention a few of these elicited
responses. Feeding by the white pine weevil (Pissodes strobi) on Sitka spruce
(P. sitchensis) induced traumatic resin accumulation in stems and also induced
expression of several terpene synthase transcripts, such as the ()-pinene synthase
(Byun McKay et al. 2003; Miller et al. 2005). In addition, ()-linalool synthase
activity increased after weevil feeding, resulting in linalool emissions (Miller
et al. 2005). Analogously, monoterpene synthase activities in needles of ponderosa
pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and white fir (Abies
concolor) were enhanced after feeding by tiger moth (Halisdota ingens) larvae
(Litvak and Monson 1998). Treatments with methyl jasmonate (MeJA), elicitor
causing responses similar to herbivore attacks, have been widely used to study
influence of biotic stress effects on terpenoid synthesis (Keeling and Bohlmann
2006b; Bohlmann 2008). In several conifers, MeJA treatment has shown to result
in formation of traumatic resin ducts, terpenoid accumulation, and induction of
prenyltransferase and terpene synthase activities, and altered volatile terpenoid
emission profiles (e.g., Martin et al. 2002, 2003; Miller et al. 2005).
There are different patterns of timing of elicitation of various terpenoid syn-
thases. In response to wounding in the gymnosperm tree Abies grandis, monoter-
pene synthase genes were elicited first, followed by sesqui- and diterpene synthases
(Steele et al. 1998). In the angiosperm tree Alnus glutinosa, feeding by com-
mon white wave (Cabera pusaria) larvae resulted in simultaneous elicitation of
emissions of mono-, sesqui and homoterpenes, but sesquiterpene emissions were
characterized by biphasic emission kinetics and the level of elicitation differed for
different volatile terpenes (Copolovici et al. 2011). Such variations in temporal
patterns can be simulated based on the theory of recursive action of regulators
on the target gene(s) over time (Vu and Vohradsky 2007) as demonstrated in the
80 H. Rajabi Memari et al.

chapter of Grote et al. (2013). In addition, there are also genotypic differences in the
degree of elicitation of given synthases (Byun-McKay et al. 2006), suggesting that
modifications in gene regulation patterns can constitute a further important adaptive
mechanism.
Overall, the phenomenological evidence consistently indicates the biotic stress-
dependent enhancement of terpenoid synthesis pathway and modifications of
terpenoid profiles. Although there has been a significant progress in biochemical
and molecular characterization of the induced terpenoid responses (Huber et al.
2004; Martin and Bohlmann 2005; Keeling and Bohlmann 2006b; Tholl 2006; Chen
et al. 2011), regulation of stress-induced terpene synthesis is still poorly known.
In white spruce (Picea glauca), putative cis-acting elements such as MeJA- and
wound-response elements, and promoter-enhancing sequences have been identified
for monoterpene 3-carene synthase by BAC cloning (Hamberger et al. 2009), but
the inducible terpenoid promoters have not yet been functionally tested. Potato
(Solanum tuberosum) wound- and insect-inducible promoter of proteinase inhibitor
protein was used to demonstrate local elicitation of terpenoid synthase activity in
transgenic Picea glauca after mechanical wounding (Godard et al. 2007). This
transgenic system provides encouraging platform for proof-of-concept studies, and
possibly will also help to elucidate the regulation of inducible terpenoid synthases
in native systems.

3.6 Conclusions

More than 60,000 terpenoid compounds have been described in living organisms.
This huge chemical diversity results from a large number of terpenoid synthases
and relatively low product specificity of many terpenoid synthases. High genetic
richness of terpenoid synthases present in many plant species constitutes an
important gene pool for adaptation. Especially, given that the product profiles of
several terpenoid synthases can be altered with only minor modifications in active
center structure, gene duplication of ancient progenitor terpenoid synthases has
catalyzed the vast evolutionary adjustment in terpenoid profiles (Sect. 3.4.2). On
the other hand, millions years of evolution have led to low sequence similarity
among terpenoid synthases in distant organisms. However, sequence similarity is not
necessarily associated with protein function, as the classic class I and class II terpene
synthase tertiary structures are remarkably similar even across the domains of life.
In fact, widely divergent terpene synthases at sequence level can make the same
products and have similar product profiles. Thus, there is evidence of convergent
evolution in terpenoid synthases among many plant groups.
So far, terpenoid synthase gene family structure is available only for a few tree
species, with particularly limited information for key forest species, albeit highly
detailed information has been gained by next generation transcriptome sequencing
techniques and several full genome sequences for important tree species have
become available (Sect. 3.4.2). As more genomic data become available, we will be
3 The Biochemistry and Molecular Biology of Volatile Messengers in Trees 81

able to gain more conclusive insight into the variations in terpenoid synthase gene
family size and diversity. Even among the sequenced organisms, recent evidence of
polyphyletic origin of terpenoid synthases has been found, suggesting that genetic
diversity of plant terpenoid synthases may be even larger than we have thought
before (Li et al. 2012).
There is rich phenomenological evidence of changes in constitutively expressed
synthase activities and induction of non-constitutively expressed terpenoid syn-
thases by different abiotic and biotic stresses for many tree species (Sect. 3.5).
However, the information on regulatory elements of terpenoid synthases is currently
very limited, and clearly more work is needed to gain insight into regulation of the
expression of terpenoid synthases as driven by environmental variability and stress.

Acknowledgements Authors’ work on plant volatiles is funded by the Estonian Ministry of

Science and Education (institutional grant IUT-8-3), the European Science Foundation (Eurocores
project A-BIO-VOC), the European Commission through European Regional Fund (the Center
of Excellence in Environmental Adaptation) and the European Research Council (advanced
grant 322603, SIP-VOLC). We thank Dr. Maaria Rosenkranz for insightful comments on the
manuscript.

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Chapter 4
Genetic Engineering of BVOC Emissions
from Trees

Maaria Rosenkranz and Jörg-Peter Schnitzler

Abstract In recent years, it has become possible to inhibit constitutive production

of biogenic volatile organic compounds (BVOC) in trees by genetic engineering.
In addition, the trait for constitutive emissions has been introduced in several
previously non-emitting herbaceous model organisms and crops. Research on these
genetically engineered organisms has demonstrated that eliminating terpenes (syn.
terpenoids or isoprenoids) emission reduces stress tolerance, while enhancing
emissions often increases abiotic and biotic stress tolerance. In this chapter, the
progress in terpene engineering work is reviewed, and the advantages of, changes in
and obstacles related to genetically modified (GM) trees are discussed. We start
by introducing the reader to terpene biosynthesis and the efforts undertaken to
manipulate that process, we further review past attempts to repress and overexpress
terpene synthases in herbs and trees, and finally describe the current achievements
and suggest future possibilities in the field of terpene emission engineering.

4.1 Introduction

During the last few decades, it has become possible to transfer specific traits into dif-
ferent organisms, silence the expression of specific genes or analyse tissue-specific
activation and regulation of promoter elements. The first genetically modified (GM)
plant was a tobacco (Nicotiana tabacum) with a chimeric antibiotic resistance gene
(Bevan et al. 1983). A few years later, the first GM tree, a hybrid poplar (Populus
alba x P. grandidentata) carrying a gene for herbicide resistance, was developed
(Fillatti et al. 1987). Genetic modifications have the potential for a wide range of
applications (Leroy et al. 2000). GM trees have multiple socioeconomic benefits,

M. Rosenkranz () • J.-P. Schnitzler

Research Unit Environmental Simulation (EUS), Institute of Biochemical Plant Pathology,
Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany
e-mail: [email protected]

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 95
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 4,
© Springer ScienceCBusiness Media Dordrecht 2013
96 M. Rosenkranz and J.-P. Schnitzler

including faster growth (Kawaoka et al. 2003; Jing et al. 2004), resistance to cold
(Jin et al. 2009), herbivorous insects (Wang et al. 1996), and viruses (Gonsalves
et al. 2004), as well as soil phytoremediation (Peuke and Rennenberg 2005). Given
the central role of wood pulping and paper production, one of the main foci of GM
tree research involves altering the metabolic flux of lignin biosynthesis pathways
(Chen and Dixon 2007; Jouanin and Lapierre 2012; Meyermans et al. 2000; Pilate
et al. 2004). Trees with reduced lignocellulose content need less extensive treatment
with hot sulfuric acid during pulp production, and therefore reduce energy inputs,
and the use of chemicals and white-water pollution (Herschbach and Kopriva 2002).
GM trees carry particular traits that make them novel to ecosystems (e.g., com-
mercially available Bt-poplars in China (Wang 2004) or papaya (Carica papaya)
trees resistant to papaya ringspot virus (PRVS) in Hawaii (Gonsalves et al. 2004)).
The potential impacts of GM trees on native ecosystems are mainly ecological,
as these trees could influence the fitness of other species, population dynamics,
ecological roles, interspecies interactions, and air chemistry.
Because forests – the “lungs” of the Earth – consume CO2 and other gases, they
occupy a central role in maintaining tropospheric chemistry (Schulze and Caldwell
1995). In addition to oxygen, forests emit a wide variety of biogenic volatile organic
compounds (BVOCs). Forests are globally the major source of BVOC emissions
(Boissard et al. 2001). Isoprene (worldwide 44 % of total emissions) dominates
BVOC emissions (Guenther et al. 1995). Due to its high reactive capacity in the
atmosphere, it strongly modifies atmospheric reactivity, in particular, causing ozone
formation together with NOx (Fuentes et al. 2000). Monoterpenes (11 %) constitute
the second most important reactive class of BVOCs emitted from plants (Guenther
et al. 1995; Fuentes et al. 2000). Moreover, the temperate and tropical forests
are known to emit approximately 60 different chemicals in significant quantities
(Kesselmeier and Staudt 1999), making it especially difficult to target specific
modulations of BVOC emissions. It is unfortunate that most tree species used
in agroforest plantations, including poplars (Populus spp.), willows (Salix spp.),
eucalypts (Eucalyptus spp.) and oil palm (Elaeis guineensis) emit BVOCs, and
particularly isoprene, in large quantities (Kesselmeier and Staudt 1999; Owen et al.
2013). These emissions tend to promote the production of atmospheric oxidants in
urban and suburban regions (Chameides et al. 1988; Owen et al. 2013), especially in
atmospheres with abundant NOx due to human activities (Hewitt et al. 2009). Thus,
from an air quality perspective, low or non isoprene-emitting trees are desirable, and
a reduction in the isoprene emissions from these trees would decrease the isoprene
fluxes from plantations.
In addition to the efforts to modify tree wood quality, growth and resistance
to stress, there are also attempts to create novel emission profiles in plants. GM
plants with modified emission profiles (mainly with regard to terpenoids) are widely
used to investigate plant-insect and plant-microbe interactions (e.g., Gershenzon and
Dudareva 2007) and analyse ‘upstream’ metabolic processes, e.g., regulation of the
mevalonate (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-
xylulose 5-phosphate pathway (MEP/DOXP pathway). Different approaches can be
used to add or remove genes and enhance or decrease the existing emission activity.
4 Genetic Engineering of BVOC Emissions from Trees 97

Plants with engineered emission profiles will help to elucidate the physiological
and ecological roles of these compounds. In addition, healthier plants with better
resistance to environmental stresses or plants producing economically important
compounds, e.g., pharmaceuticals, can be generated (Chang and Keasling 2006;
Bouwmeester 2006).
There have been several attempts to modify terpene emissions in plants (e.g.,
Behnke et al. 2007; Kappers et al. 2005; Laothawornkitkul et al. 2008; Vickers et al.
2009; Lee et al. 2010). One of the main obstacles is the complex terpene biosyn-
thesis regulatory system, which is not well understood. The successful modification
of BVOC emissions requires an understanding of the different regulatory steps and
feedback mechanisms in these pathways, and as these mechanisms differ between
species, engineering work is even more challenging.
The biochemical terpene-world is complex; there are several different com-
pounds with diverse structures (Gershenzon and Dudareva 2007). All of these
compounds, including the non-volatile terpenes are produced from key interme-
diate compounds, isopentenyl diphosphate (IDP) and dimethylallyl diphosphate
(DMADP) (McCaskill and Croteau 1998; Lichtenthaler 1999). Thus, metabolic
elements downstream of the initial substrate are necessary to regulate the synthesis
of different terpene compounds. More than once, the modification of a non-
emitting species towards increased emissions has failed because there was not
enough precursor substrate to produce moderate to high emission levels (Kappers
et al. 2005; Sharkey et al. 2005; Schnee et al. 2006; Loivamäki et al. 2007a, b;
Sasaki et al. 2007). Thus, increasing the substrate availability for terpene biosyn-
thesis by boosting the terpene production pathway has provided promising results
(Wu et al. 2006).
In this chapter, we first introduce the biosynthetic pathways for terpene pro-
duction. We will give an overview of the efforts to engineer terpene-producing
pathways that modify substrate availability and/or help to elucidate the pathway
regulation systems. We continue by discussing the different approaches to the
genetic engineering of BVOC profiles. Studies investigating the transcriptional
regulation of emissions are discussed, and we outline the current work on the
engineering of terpene emission profiles in trees and herbs. We concentrate on
isoprene, the compound dominating the global BVOC emissions, but we also
cover mono- and sesquiterpene emission engineering. Finally, we discuss the use
of reporter lines that allow for monitoring activation of pathways and location of
enzymes in BVOC studies.

4.2 Overview of BVOC Biosynthesis Pathways

with Emphasis on Genetic Engineering

BVOC biosynthesis, regulatory pathways and emissions are complex. One of the
problems related to genetically engineering BVOC emissions is substrate avail-
ability. Plant terpenoids are synthesized through condensation of the five-carbon
98 M. Rosenkranz and J.-P. Schnitzler

precursors IDP and DMADP, which can be synthesized in two pathways localized
in two separate cell compartments. The availability of precursors is an important
issue in order to succeed in gene engineering.
Kinetic characteristics of the two terpenoid synthesis pathway are discussed
in Li and Sharkey (2013). In the present chapter the two pathways and the
metabolic cross-talk between them are briefly reviewed for the purpose of a global
understanding of the complex regulation of the emission profiles. This is done
mainly by discussing the various attempts to genetically engineer or chemically
manipulate flux through the pathways in order to gain better understanding of the
regulation processes and analyse what is specific in plant species with high rate
of terpene synthesis compared with plants with low terpene synthesis rate. Also
the recently discovered terpene precursor biosynthesis pathway in mitochondria is
discussed. Although volatile terpenoids are often considered secondary metabolites,
many terpenoids also participate in primary metabolism, including plant hormones
gibberellins, cytokinins, and abscisic acid and components of the photosynthetic
machinery such as plastoquinol, phytol residue of chlorophyll and carotenoids.
Thus, it is often difficult to separate “primary” and “secondary” metabolism
(Fineschi et al. 2013), further emphasizing the difficulties with engineering terpene
metabolism.

4.2.1 Cytosolic Terpene Production

The cytosolic mevalonate (MVA) or 3-hydroxy-3-methylglutaryl coenzyme A

(HMG-CoA) pathway is well understood (Agranoff et al. 1960). This pathway is
required for the synthesis of cytokinins, sesquiterpenes (C15), and triterpenes (C30,
e.g., sterols and brassinosteroids) (Suzuki et al. 2004). This pathway also provides
precursors for ubiquinone synthesis in mitochondria (Disch et al. 1998). The MVA
pathway is found in most eukaryotic organisms including animals, fungi and plants,
but also in Archaea and in some bacteria (Lombard and Moreira 2011; Chappell
et al. 1995). The MVA pathway starts with production of acetoacetyl-CoA from two
molecules of acetyl-CoA. This step is catalyzed by the enzyme AcAc-CoA thiolase
2 (AACT2) (Vollack 1996; Ahumada et al. 2008). In the second step, HMG-CoA
synthase condenses acetyl-CoA and AcAc-CoA to form HMG-CoA. The next step
is one of the most studied enzymatic reactions in terpene biosynthesis (McCaskill
and Croteau 1998; Rodrı́guez-Concepción 2006). HMG-CoA reductase catalyses
the synthesis of mevalonic acid from HMG-CoA. The HMG-CoA reductase is
encoded by a multigene family whose size varies from plant to plant (Stermer
et al. 1994), and its expression is tightly controlled at the transcriptional and post-
transcriptional levels (reviewed in Rodrı́guez-Concepción 2006). The HMG-CoA
reductase can be activated by multiple different promoters (Lumbreras et al. 1995).
This complex regulatory system underlines the importance of the enzyme. HMG-
CoA reductase controls the flux of the MVA pathway (Rodrı́guez-Concepción 2006)
4 Genetic Engineering of BVOC Emissions from Trees 99

and, consequently, it is also the cytosolic enzyme upstream from IDP that has been
targeted to gene engineering in order to enhance terpene emissions (Chappell et al.
1995). Overexpression of the gene in tobacco (Nicotiana tabacum) did, however,
not alter the sesquiterpene emission, but sterol levels were enhanced (Rodrı́guez-
Concepción 2006). HMG-CoA can be, in addition, chemically blocked by mevinolin
or lovastatin. By this means the exchange of precursors between the cytosolic MVA
and chloroplastic MEP pathways can be examined (e.g., Rodrı́guez-Concepción
et al. 2004; Laule et al. 2003) (see below for more information about the exchange).
In the next step, mevalonate kinase phosphorylates mevalonate to form meval-
onate 5-phosphate, which is further phosphorylated by phosphomevalonate kinase
to mevalonate 5-diphosphate. Finally, mevalonate diphosphate carboxylase cataly-
ses the decarboxylation of mevalonate 5-diphosphate to IDP. These last steps are
well studied in yeast (Saccharomyces cerevisiae) and mammals but not yet in plants
(Nagegowda 2010).
Isopentenyl diphosphate isomerase (IDI) is an enzyme that forms DMADP
from IDP and vice versa, and this reaction can occur in both cytosol and plastids
(Ramos-Valdivia et al. 1997; Brüggemann and Schnitzler 2002a), albeit the transfer
of isoprenoid precursors among compartments (and among pathways) can be
limited (Sect. 4.2.3). DMADP is the direct precursor of the hemiterpene isoprene
in chloroplasts, whereas IDP is used to form higher terpene precursors. For
higher terpene synthesis, the two diphosphorylated C5-units, DMADP and IDP,
condense in a head-to-tail reaction to produce geranyl diphosphate (GDP; C10),
which is the immediate precursor of monoterpenes. Condensation of GDP and
IDP produces farnesyl diphosphate (FDP; C15), which is an immediate precursor
of sesquiterpenes, while condensation of FDP and IDP results in formation of
geranylgeranyl diphosphate (GGDP; C20), a diterpene precursor (Ramos-Valdivia
et al. 1997).
Chen et al. (2000) showed that overexpression of FDP in sweet sagewort
(Artemisia annua L.) can lead to a slightly higher production of the sesquiterpene
artemisinin. More recently Wu et al. (2006) achieved high sesquiterpene emission
levels by introducing a novel construct, in which an avian FDP synthase was
combined with the sesquiterpene patchoulol synthase, into tobacco plants. Although
sesquiterpenes are primarily synthesized via the MVA pathway, small sesquiterpene
emissions have been reported also from plastidic FDP pools (Aharoni et al. 2003;
Wu et al. 2006), and sesquiterpene synthesis could be engineered by this way both
to cytosolic and chloroplastic compartments of tobacco (Wu et al. 2006).

4.2.2 Plastidic Terpenoid Production

DMADP in chloroplasts is synthesized through the MEP/DOXP pathway (Rohmer

et al. 1993; Lichtenthaler et al. 1997; Fig. 4.1; Phillips et al. 2008). Previously, it was
assumed that all isoprenoids are synthesized exclusively through the MVA pathway,
but by now, the MEP pathway has also been found in Eubacteria, Chromalveolata,
100 M. Rosenkranz and J.-P. Schnitzler

Fig. 4.1 Diagram of the plastidic 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose

5-phosphate (MEP/DOXP) pathway for isoprenoid synthesis. The possible regulatory steps, shown
to correlate with the end-product level, are marked red. The individual steps are discussed in
the Sect. 4.2.2. The enzymes catalyzing the reactions are shown in bold font as follows: 1-
deoxy-D-xylulose 5-phosphate synthase (DXS); 1-deoxy-D-xylulose 5-preductoisomerase (DXR);
2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT); 4-(cytidine 5’-diphospho)-2-
C-methyl-D-erythritol kinase (CMK); 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase
(MDS); 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS), 4-hydroxy-3-methylbut-2-
enyl diphosphate reductase (HDR); isopentenyl diphosphate isomerase (IDI); isoprene synthase
(IspS). Shown are also ways of chemical manipulation of the pathway by e.g., blocking with
fosmidomycin (FSM) or feeding with di-deuterated deoxyxylulose (DOX)

and algae (Eoh et al. 2007; Cassera et al. 2004; Grauvogel and Petersen 2007;
Okada and Hase 2005; Massé et al. 2004). This pathway produces IDP and its
isomer DMADP, which are the building units of isoprene (C5), monoterpenes (C10),
diterpenes (C20), carotenoids (C40), phytol, tocopherols, phylloquinones, and some
sesquiterpene (C15) (Lichtenthaler 1999). In isoprene emitters, the major product
of the MEP pathway goes into isoprene emission (Wolfertz et al. 2004; Li and
Sharkey 2013), but the importance of the production of higher terpenoids should
not be underestimated.
Terpene products from the MEP pathway are directly coupled to photosynthesis
through the C3 metabolites glyceraldehyde 3-phosphate (GAP) and pyruvate that
are the substrates for the first MEP pathway reaction. In this first step of the
MEP pathway, a C2 unit from pyruvate is transferred to GAP by 1-deoxy-D-
4 Genetic Engineering of BVOC Emissions from Trees 101

xylulose 5-phosphate synthase (DXS) forming 1-deoxy-D-xylulose 5-phosphate

(Lichtenthaler 1999). GAP is a product of the Calvin cycle, but the source of
pyruvate in chloroplasts is not fully clear. Chloroplasts may import phosphoenol
pyruvate (PEP) from cytosolic glycolysis that is further converted to pyruvate (Affek
and Yakir 2003; Trowbridge et al. 2012), but pyruvate might also originate from
chloroplastic metabolism (Sharkey and Yeh 2001; Rasulov et al. 2011; Li and
Sharkey 2013). Under certain environmental conditions, carbon from stored sources
can be mobilized to keep the MEP pathway active (Schnitzler et al. 2004).
DXS as the first enzyme in the MEP pathway may have a regulatory role, as its
expression positively correlates with the concentration of several isoprenoid end
products (Lois et al. 2000; Estévez et al. 2001; Muñoz-Bertomeu et al. 2006).
Estévez et al. (2001) showed that overexpression of DXS in Arabidopsis thaliana
leads to a moderate increase of carotenoid and abscisic acid (ABA) levels. Similar
results were obtained for tomato (Lycopersicon esculentum) (Lois et al. 2000)
and in lavender (Lavandula latifolia) (Muñoz-Bertomeu et al. 2006). DXS forms
1-deoxy-D-xylulose 5-phosphate (DOXP), which is converted to MEP by 1-deoxy-
D-xylulose 5-phosphate reductoisomerase (DXR). DOXP is not only the first
intermediate of the MEP pathway, but it is also involved in the thiamine/shikimate
pathway (Julliard and Douce 1991; Belanger et al. 1995). It has been assumed that
DXR rather than DXS plays a regulatory role in the MEP pathway (Mayrhofer
et al. 2005). The transformation of peppermint (Mentha x piperita) to enhance
DXR activity increased the plant’s essential oil yield (Mahmoud and Croteau 2001).
Moreover, Carretero-Paulet et al. (2002; 2006) showed that overexpression of DXR
in Arabidopsis thaliana increased the concentration of isoprenoid end-products.
Additionally, Mahmoud and Croteau (2001) showed that altered DXR levels alter
the flow through the MEP pathway in Arabidopsis, which supports the role of
DXR as a regulatory step. Because DXR gene expression does not correlate with
the rhythm of isoprene emission (Mayrhofer et al. 2005; Loivamäki et al. 2007b),
the extent of its regulatory role remains unknown. On the other hand, reports by
Estévez et al. (2001) and Enfissi et al. (2005) on tomato plants with altered DXS
expression strongly suggested that DXS is the main regulator of the MEP pathway.
These authors proved that changes in DXS levels result in similarly changed end-
product profiles (Estévez et al. 2001; Enfissi et al. 2005). Feeding with an external
substrate, di-deuterated deoxyxylulose (DOX) produces similar results (Loivamäki
et al. 2007a). DOX is rapidly incorporated into the MEP pathway and can be used
downstream from DXS to enhance the flow through the pathway (Fig. 4.1). In
Eucalyptus globulus, however, isoprene emission was not changed by DOX-feeding,
indicating that some intermediates of the MEP pathway contribute to a negative
feedback loop (Wolfertz et al. 2004; Monson 2013).
The MEP pathway can be blocked with fosmidomycin, a DXR reductoisomerase
inhibitor (Kuzyama et al. 1998; Fig. 4.1). Fosmidomycin has been used to study dif-
ferent aspects of isoprene biosynthesis and its physiological significance (Sharkey
et al. 2001; Loreto and Velikova 2001; Velikova and Loreto 2005; Rasulov et al.
2009a; Rasulov et al. 2011). Because the inhibitor is not specific to isoprene and
monoterpene biosynthesis - it also decreases synthesis of carotenoids and other
102 M. Rosenkranz and J.-P. Schnitzler

MEP pathway end-products – longer-term changes in plant physiological status after

suppression of isoprene emission by fosmidomycin-feeding (e.g., altered response
to heat or oxidative stress, Possell and Loreto 2013) cannot be exclusively attributed
to isoprene depletion. Recent study showed that fosmidomycin treatment could lead
to higher susceptibility to photoinhibition and photodamage at high light intensities
(Possell et al. 2010), which might be partly due to inhibition of biosynthesis of
carotenoids.
In the next steps of plastidic terpenoid synthesis, 2-C-methyl-D-erythritol 4-
phosphate cytidylyltransferase (MCT) catalyzes the production of 4-(cytidine
50 -diphospho)-2-C-methyl-D-erythritol (Eisenreich et al. 2001; Phillips et al.
2008). This intermediate is further converted to 2-C-methyl-D-erythritol 2,4-
cyclodiphosphate by 4-(cytidine 50 -diphospho)-2-C-methyl-D-erythritol kinase
(CMK) and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS). In the
next step, 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS) converts
2-C-methyl-D-erythritol 2,4-cyclodiphosphate to 4-hydroxy-3-methylbut-2-enyl
diphosphate. The last step in the MEP pathway is catalyzed by the enzyme
4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), which converts 4-
hydroxy-3-methylbut-2-enyl diphosphate to DMADP and IDP. HDR may have an
additional regulatory role in the MEP pathway, as its enhanced expression results
in higher carotenoid concentrations in Arabidopsis (Botella-Pavı́a et al. 2004).
Thus, this evidence suggests that plastidic terpenoid production may be regulated at
various points in the pathway (Fig. 4.1).
Wiberley et al. (2009) cloned all the genes from Populus trichocarpa and
measured their expression levels under various environmental conditions. They
showed that the DXS and HDR genes are regulated by circadian rhythms and the
expression of DXS, DXR and CMK genes was temperature-dependent (Wiberley
et al. 2009), similarly to the regulation of the isoprene synthase (IspS) gene
(Loivamäki et al. 2007a, Sect. 4.3.1). Thus, multiple enzymes, including DXS, could
have a regulatory role in the MEP pathway of P. trichocarpa (Wiberley et al. 2009).
As in the MVA pathway (Sect. 4.2.1), chloroplastic IDI converts IDP (the
precursor of higher terpenoids) to DMADP (the immediate precursor of isoprene)
and vice versa. IspS converts chloroplastic DMADP to isoprene. In addition to
isoprene biosynthesis, monoterpene biosynthesis takes place in chloroplasts. GDP
that is formed by head-to-tail condensation of IDP and DMADP is the precursor
for various monoterpenes, whose synthesis is catalyzed by different monoterpene
synthases (Lichtenthaler 1999), each of which often synthesizes multiple monoter-
pene species (Tholl 2006; Rajabi Memari et al. 2013). The regulation of emissions
at transcription level is discussed in Sect 4.3 (see also Monson 2013) and by
environmental drivers in other chapters of this book (Grote et al. 2013; Li and
Sharkey 2013; Monson 2013).
Terpene precursors are present in chloroplasts and mitochondria. Kappers et al.
(2005) targeted an Arabidopsis terpene synthase (Tps) to mitochondria and mea-
sured low amounts of nerolidol and linalool, which are terpenes that do not naturally
occur in Arabidopsis BVOC emissions. Because FDP is present in mitochondria,
the introduced sesquiterpene synthase resulted in terpene emission. The presence
4 Genetic Engineering of BVOC Emissions from Trees 103

of FDP could be expected because the cytosolic IDP precursor is transported to

mitochondria, where it is utilised for ubiquinone biosynthesis (Disch et al. 1998).
It is still unclear whether terpene precursors have functions in the mitochondria in
other plant species.

4.2.3 Interactions Between MEP and MVA Pathways

The interactions between the plastidic MEP and cytosolic MVA pathways also need
clarification (Lichtenthaler 1999; Laule et al. 2003; Rodrı́guez-Concepción et al.
2004). There may be a cross-talk between the two pathways in some species and
plant tissues, such that MVA derived precursors can be used in plastids for iso-
prenoid biosynthesis and vice versa (Lichtenthaler 1999). Laule et al. (2003) showed
that blocking the MVA pathway with lovastatin causes changes in chloroplastic
carotenoid and chlorophyll contents and in transcript levels of the rate-controlling
enzymes, whereas cytosolic sterol levels remained constant after recovering from
an initial drop. The authors suggested that the transport of IDP from chloroplast to
cytosol was responsible for these effects.
Blocking the MEP pathway with fosmidomycin resulted in decreased concen-
trations of all the terpenoid metabolites and the cytosolic sterols (Kuzyama et al.
1998). Taken together with the data from Laule et al. (2003), these results suggest
unidirectional transport of IDP from chloroplasts to the cytosol. This hypothesis
is supported by work from Loreto et al. (2004), who used 13 C-labelling and
fosmidomycin to show that a possible cross-talk between MEP and MVA pathways
does not modulate isoprene emission levels when the MEP pathway is blocked
at least for the time-period of the experiment. A certain recovery of isoprene
emissions after fosmidomycin inhibition can be sometimes observed in longer-term
experiments (Rasulov et al. 2011).
In contrast to these findings, Rodrı́guez-Concepción et al. (2004) isolated
Arabidopsis mutants that survived when the MEP pathway was blocked. This result
may have been due to enhanced uptake of MVA derived terpenoid precursors by
plastids, but no definitive proof of an enhanced cytosol-to-plastid transport exists to
our knowledge.

4.3 The Regulation of Emissions During and After

Transcription

4.3.1 Isoprene Synthase

Before the discovery of IspS (Silver and Fall 1991), isoprene formation was thought
to occur spontaneously, as the two phosphate residues can be lost and a double bond
104 M. Rosenkranz and J.-P. Schnitzler

formed at low rates in acidic conditions. The relevance of IspS in isoprene emissions
was demonstrated in several species by the positive correlation between the enzyme
activity and basal emissions (Lehning et al. 1999; Brüggemann and Schnitzler
2002c; Kuzma and Fall 1993; Scholefield et al. 2004; Mayrhofer et al. 2005).
IspS can exist in two isoforms. The soluble form is located in the chloroplast
stroma, and the insoluble form is bound to thylakoid membranes, as observed in
willow (Salix discolor) by Wildermuth and Fall (1996, 1998) and in other species
(Wiberley et al. 2005; Schnitzler et al. 2005). All deduced IspS proteins have a
transit peptide that targets them to chloroplasts, where isoprene synthesis takes
place (Mgalobilishvili et al. 1979; Schnitzler et al. 2005). Most of the known plant
IspS genes have been isolated from poplar species (Miller et al. 2001; Sasaki et al.
2005; Fortunati et al. 2008; Sharkey et al. 2005; Calfapietra et al. 2007). In addition,
the presence of a IspS gene has been verified in Eucalyptus globulus, Melaleuca
alternifolia and Pueraria montana (Sharkey 2013; Li and Sharkey 2013). Sharkey
(2013) recently constructed a phylogenetic tree with all of the known angiosperm
IspS genes, suggesting that they form a monophyletic clade within the rosids. This
result suggests a single evolutionary event for IspS gene formation followed by
recurrent losses of isoprene emission capacity within this group. However, there
is evidence that the evolution of isoprene production capacity and loss has taken
place independently in various taxonomic groups of higher plants, especially if one
considers also mosses, ferns and gymnosperms (Monson et al. 2013).
Promoter studies elucidate gene regulation at the transcription level. Knowing the
promoter region sequence, regulatory elements can be predicted by in silico studies.
For example, computational analysis of the P. x canescens isoprene synthase gene
promoter revealed many putative light responsive boxes, heat shock and circadian
elements (Loivamäki et al. 2007b). Similar elements were also present in the
promoter sequence of P. trichocarpa IspS gene (Sharkey et al. 2008). Even under
constant light, IspS gene transcripts fluctuate diurnally, demonstrating the circadian
clock’s involvement in their regulation (Loivamäki et al. 2007b).
To study the gene activation in vivo, Cinege et al. (2009) cloned the IspS gene
promoter upstream of two reporter genes, green fluorescence protein (E-GFP) and
a “-glucuronidase (GUS). Analysis of these reporters and the PcIspS promoter in
Arabidopsis and poplar confirmed that the IspS gene in P. x canescens is activated
by light and elevated temperature (Cinege et al. 2009). This example demonstrates
the usefulness of GM trees for studying gene activation in real time. The circadian
regulation of isoprene emission from oil palm (Elaeis guineensis) and grey poplar
(P. x canescens) was also demonstrated (Wilkinson et al. 2006; Loivamäki et al.
2007b). Moreover, the correlation between mean air temperature and IspS gene
transcript levels was observed in P. x canescens during the vegetation period
(Mayrhofer et al. 2005).
During leaf development, isoprene emission is transcriptionally controlled. IspS
mRNA and protein appear at the same developmental stage, which can be days
to weeks after the onset of photosynthesis depending on the temperature at which
the leaves have developed (Mayrhofer et al. 2005; Wiberley et al. 2005). Similarly,
the regulation of isoprene emission through IspS transcription and IspS protein
4 Genetic Engineering of BVOC Emissions from Trees 105

levels may take place under stress, as has been observed in quaking aspen (Populus
tremuloides) grown under elevated [O3 ] (Calfapietra et al. 2007, 2008, 2013).
Several regulatory steps other than gene transcription can adjust the availability
of functional protein. While the potential post-transcriptional and post-translational
modifications of terpenes are still unclear, there have been a few demonstrations
of these types of regulation. Transcript and protein levels, enzyme activity and
isoprene emission do not always correspond on a diurnal scale (Loivamäki et al.
2007b), during a period of a few weeks (Wiberley et al. 2009) or over the course
of the growing season (Mayrhofer et al. 2005). IspS gene transcription in P. x
canescens leaves reaches its maximum in summer (July, beginning of August),
and maximal protein expression occurs in late summer and autumn. However, IspS
activity drops dramatically at the end of September, suggesting a possible post-
translational regulation of IspS (Mayrhofer et al. 2005). Similarly, the drought stress
response affects IspS activity more than its protein or transcript levels, suggesting
that there is tighter post-transcriptional control of isoprene emissions during these
conditions (Brilli et al. 2007; Fortunati et al. 2008; Laothawornkitkul et al. 2008).
The nature of the possible modifications is, however, not yet known. On the other
hand, stress conditions and aging are also known to reduce the concentrations
of DMADP further possibly reducing the emissions (Sun et al. 2012b; Li and
Sharkey 2013).

4.3.2 Other Volatile Terpene Synthases

Terpenes are the largest group of plant natural products and are structurally very
complex (Degenhardt et al. 2009). In the present chapter, we cannot include all of
the reaction mechanisms and regulatory events that are responsible for the gene
activation and biochemical regulation of monoterpene and sesquiterpene synthase
activities. The recent reviews of these topics are very comprehensive (see e.g., Tholl
2006; Christianson 2006; Degenhardt et al. 2009; Rajabi Memari et al. 2013). While
many terpene synthases (Tps) catalyze the formation of multiple products, some are
very specific in generating only one hydrocarbon product (Tholl 2006; Christianson
2006; Rajabi Memari et al. 2013). Volatile terpenes are often emitted from very
specific plant parts, such as flowers (Dudareva et al. 2003; Chen et al. 2003), fruits
(Lücker et al. 2004), young leaves (Brilli et al. 2009) or roots (Chen et al. 2004),
depending their function in plant metabolism.
Intracellular gene activation analyses in GM trees are possible with confocal laser
scanning microscopy, which clarify the roles of various genes in specific cells and
tissues. A good demonstration of this method is the work by Tholl et al. (2005),
who showed detailed activation of specific Tps gene promoters in different parts
of Arabidopsis flowers. For example, the At5g23960 terpene gene is active in the
stigma. The same authors showed that “-caryophyllene, the dominant product of
At5g23960, protects the flowers from pathogens (Huang et al. 2012). This result
demonstrated nicely that low-level, directed terpene emission is very efficient.
106 M. Rosenkranz and J.-P. Schnitzler

4.4 Genetic Engineering of BVOC Emissions – Present

Aspects and Future Potentials

4.4.1 Overexpression of Terpene Synthases

4.4.1.1 Overexpression with Constitutive Promoters

Because many Tps genes are cloned and available for further research, there are
new opportunities for engineering biogenic BVOC emissions (for a comprehensive
list of isolated monoterpene and sesquiterpene synthases, see Degenhardt et al.
2009). However, overexpressing Tps genes is more difficult than expected. Although
several publications describe a successful introduction of a new Tps gene to a plant,
almost as many papers report only very low increases in terpene emissions (Hohn
and Ohlrogge 1991; Zook et al. 1996; Aharoni et al. 2003; Kappers et al. 2005;
Loivamäki et al. 2007a; Sasaki et al. 2007). The emissions have been so low that
they hardly passed the technical limits of detection. Most of the work has been
performed in Arabidopsis, and the problem may be the scarce substrate availability
or the tight control of the substrate supply in this model organism (Aharoni et al.
2003; Besumbes et al. 2004; Loivamäki et al. 2007a).
There have been several attempts to transform Arabidopsis thaliana to an
isoprene emitting plant (Sharkey et al. 2005; Loivamäki et al. 2007a; Sasaki et al.
2007; Fig. 4.2). In these studies, different isoprene synthase genes were used
for transformation, but none resulted in high isoprene emission. As demonstrated
by Loivamäki et al. (2007a), substrate scarcity might inhibit isoprene emissions.
Because of its low emissions, Arabidopsis was, for long time thought to be
completely incapable of volatile emissions (Van Poecke et al. 2001) or to have only
a low capacity for terpene synthesis. However, even low terpene emissions can have
ecological impacts, like demonstrated for natural Arabidopsis BVOC emissions by
Van Poecke et al. (2001). Similarly, low emissions achieved by genetic engineering
can also lead to ecophysiological changes. Loivamäki et al. (2008) showed that low
isoprene emissions in Arabidopsis change the behavior of a parasitic wasp searching
for its host. Similarly, Kappers et al. (2005), who targeted nerolidol/linalool synthase
to Arabidopsis mitochondria, and Schnee et al. (2006), who introduced maize
sesquiterpene synthase into Arabidopsis, showed the ecological consequences of
low levels of emitted terpenes on insect behavior.
Constitutive isoprene emission could recently be introduced in silver birch
(Betula pendula) overexpressing the IspS gene from P. x canescens under the
control of the 35S promoter (Rosenkranz and Schnitzler, unpublished data, Fig. 4.2).
Compared to wild type plants, some GM lines emit significant amounts of isoprene,
making birch an interesting system to study isoprene functions, in particular, since
the first birch genome from Betula nana was published recently (Wang et al. 2012).
A clear success story in BVOC overexpression is that of Vickers et al. (2009),
who engineered tobacco (Nicotiana tabacum) to emit isoprene. These plants
allowed for verification of several hypotheses about the physiological and ecological
4 Genetic Engineering of BVOC Emissions from Trees 107

Fig. 4.2 Illustration of the tissue-cultured genetically modified silver birch (Betula pendula)
overexpressing the IspS gene from grey poplar (Populus  canescens) under the control of the 35S
promoter (a), and functional screening of birch leaves for isoprene emission (b). The screening
was performed as described in Loivamäki et al. (2007a) (Rosenkranz and Schnitzler unpublished
data)

functions of isoprene. For example, these plants were more resistant to oxidative
stress, and they showed slightly enhanced thermotolerance (Vickers et al. 2009).
Furthermore, the interactions between the plants and caterpillars of tobacco horn-
worm (Manduca sexta) were affected (Laothawornkitkul et al. 2008). The authors
had to, however, screen a huge amount of plants to find a few high isoprene emitters
(C. Vickers personal communication), which again emphasized the problems of
overexpressing IspS and Tps genes.
A potentially successful approach to engineering high terpene emissions in trees
could combine overexpression of a Tps gene and genes that regulate the MEP
or MVA pathways. This approach was used by Wu et al. (2006) who combined
FDP synthase with a sesquiterpene synthase in one construct and a GDP synthase
with monoterpene synthase in another construct in order to engeneer tobacco
plants. This work resulted in 1,000-fold higher sesquiterpene emissions and 10–
30-fold higher monoterpene emissions than in wild-type tobacco plants. In another
approach, Besumbes et al. (2004) used an inducible expression system to allow for
more efficient use of substrates. The authors transformed Arabidopsis plants with
diterpene taxadiene synthase from conifer Taxus baccata under tight regulation of
the glucocorticoid system. The terpene yield was approximately 30-fold higher than
in a constitutive expression system (Besumbes et al. 2004). This system should also
be tested in trees.
108 M. Rosenkranz and J.-P. Schnitzler

In some studies, the up-regulation of gene expression was successful in trees.

Genetic transformation has been used more often in fruit trees, principally to
enhance their disease resistance. Additionally, marker-free GM trees and improved
fruit quality are the common goals in engineering fruit trees (reviewed in Gambino
and Gribaudo 2012). Another economically interesting and thus active line of
investigation is for expansion of GM tree biomass production for use in second-
generation biofuel development. The sustainability and biomass yield of different
tree species has been enhanced by improving stress tolerance, wood properties, root
formation, or phytoremediation (Harfouche et al. 2011; Osakabe et al. 2011).
It seems more problematic to overexpress a Tps gene in a species in which
the function already exists. Thus, the approach has sometimes rather worked
against itself than created higher emission levels (Mahmoud and Croteau 2001;
Behnke et al. 2007). Behnke et al. (2007) attempted to create transgenic poplars
emitting higher amounts of isoprene, but instead, native isoprene emission was
repressed. Similarly, DXR overexpression in peppermint (Mentha x piperita L.)
led to downregulation of MEP pathway products and chlorophyll deficient plants,
indicating a co-suppression effect (Mahmoud and Croteau 2001).

4.4.1.2 Inducible Overexpression and Transient Overexpression

Most attempts to overexpress Tps genes used constitutive promoters, which might
lead to high stress and energy consumption. Native isoprene emission is strictly
regulated, and the emission level depends on many environmental conditions,
such as leaf developmental stage, time of the day and the year (Brüggemann and
Schnitzler 2002b; Kuzma and Fall 1993; Loivamäki et al. 2007b; Mayrhofer et al.
2005), plant past growth conditions (Wiberley et al. 2005; Sun et al. 2012a) and
the level of abiotic stress (Loreto and Schnitzler 2010 for a review). In Arabidopsis,
terpenes are emitted mainly from specific cells, which keeps emissions low, protects
other plant parts and limits the costs of plant metabolism (Tholl et al. 2005; Huang
et al. 2012). For BVOC emission engineering in trees, it might be interesting to use
inducible or natural promoters to study the emission profiles in these conditions.
If there were less profound changes to plant physiology, the possible effects of
overexpression of Tps genes could become clearer.
Although widely used in other areas of plant science, especially in plant
pathology, transient overexpression in herbaceous and woody plants has not been
included in BVOC studies (Chrisholm et al. 2005; Grosskinsky et al. 2011). The
short time interval and the local treatment are disadvantages that limit the use
of transient transformation for BVOC expression. We have tried to transiently
overexpress IspS gene in poplar protoplasts, but with little success (Rosenkranz
and Schnitzler unpublished). In some cases, transient transformation is used to
test the functionality of constructs (Meyer et al. 2004) before the initiation of
labor- and time-intensive procedures required to produce transgenic trees (personal
communication, A. Schmidt, MPI-ICE, Jena Germany).
4 Genetic Engineering of BVOC Emissions from Trees 109

Phenotype of isoprene emission-suppressed poplars (compared to wild type)

• Net photosynthesis sensitive to transient heat (sun) flecks

(Behnke et al. 2007; 2010a, Way et al. 2011)

• Enhanced heat dissipation as non-photochemical quenching

(Behnke et al. 2007; 2010a, Way et al. 2011)

• Enhanced ozone tolerance due to increased soluble antioxidants

(Behnke et al. 2010)

• Multiple alterations in transcript and metabolite levels

(Behnke et al. 2010b)

• Altered susceptibility to fungi and herbivorous leaf beetles

(Behnke et al. 2012)

• Slightly higher growth rates under Mid-European climate

(Behnke et al. 2012)

Fig. 4.3 Overview on phenotypic and chemotypic changes in genetically modified hybrid poplar
(Populus  canescens) due to the repression of isoprene emission

4.4.2 Repression of Terpenoid Synthases

To engineer BVOC emissions in trees, single gene knock-downs or knock-outs

may be more efficient than overexpression. In transgenic non-isoprene emitting
poplars, isoprene emissions are almost fully repressed (Behnke et al. 2007). Isoprene
emissions can be controlled by both IspS activity and substrate levels. Emissions
from immature leaves in different poplar species depend on gene activation and IspS
levels, whereas emissions in mature leaves depend both on substrate availability
and IspS activity (Ghirardo et al. 2010; Vickers et al. 2010; Rasulov et al.
2009b; Rasulov et al. 2010). Therefore, suppression of IspS could reduce isoprene
emissions from trees.
Currently, the only GM trees with reduced isoprene emission are grey poplars
(P. x canescens) (Behnke et al. 2007; Schnitzler et al. 2010). In these lines, IspS is
effectively suppressed by RNA interference (RNAi). Studies with these trees clearly
demonstrate that isoprene is important for CO2 assimilation and photosynthetic
electron transport (Behnke et al. 2007, 2010a; Way et al. 2011, Fig. 4.3). A recent
study (Behnke et al. 2012) documents the growth performance and fitness of
these GM lines over two growing seasons in ambient, near-natural conditions in
Göttingen, Germany. In the moderate climate of middle Europe, the trees’ growth
and biomass yield were not impaired but were instead temporarily enhanced in GM
isoprene-suppressed lines compared to the natural isoprene emitters. However, the
trees’ susceptibility to biotic stress (herbivores and fungi) was altered. More field
trials in various climatic and soil conditions are needed to clarify whether non-
emitting bioenergy trees can prevent negative atmospheric impacts in regions with
large wood tree plantations (e.g., Brazil, China, US, and Indonesia). A field trial with
110 M. Rosenkranz and J.-P. Schnitzler

isoprene emission-suppressed GM poplars has been started in 2012 in Oregon and

Arizona (Schnitzler, Rosenstiel, Strauss, Monson, personal communication). That
study will analyse whether the absence of isoprene emission impairs plant behavior
in more extreme climates (warm/hot and dry summer) and the natural soil conditions
of the northwestern and southwestern US.

4.5 Conclusion and Outlook

Since the first GM poplar was generated 25 years ago, numerous transformation
protocols have been established for important forest trees such as pines (Pinus
spp.), spruces (Picea spp.) and eucalypts (Eucalyptus spp.) (Harfouche et al.
2011; Osakabe et al. 2011), for commercially important fruit trees such as apple
(Malus domestica), orange (Citrus sinensis), lemon (Citrus limon), plum (Prunus
domestica), cherry (Prunus avium) and olive (Olea europaea) (Gambino and
Gribaudo 2012); as well as woody perennials like grape (Vitis vinifera) (Jin et al.
2009). Many of these species are strong emitters of terpenes (Kesselmeier and
Staudt 1999), making them interesting targets for transgenic approaches to terpene
emission modifications. Terpene emissions can be modulated by the introduction
of new Tps genes or knockdown of species-specific genes, as shown in poplar trees
(Behnke et al. 2007). Moreover, these systems might be very useful for investigating
terpenoid precursor biosynthesis via the MVA and MEP pathways in different
tissues, such as resin ducts in wood and needles. Genetic modifications of trees
could also be important for generating medically relevant terpenes. For example,
the tea tree Melaleuca alternifolia, whose bark is very rich in bioactive terpenes
and the volatiles terpinen-4-ol, ’-terpineol and 1,8-cineole (Taga et al. 2012),
would be an interesting target for genetic engineering. It is enticing to speculate
that monoterpene biosynthesis in this tree could be further enhanced by changing
the MEP pathway metabolic flux or by overexpressing the monoterpene ‘cineole
cassette’ (Faehnrich et al. 2011). Taken together, the available GM trees and the
resources for known genes of terpenes and the MVA and MEP pathways are versatile
tools that can be used to test the biological and ecological functions of volatile
terpenes in tree species that are important for forestry, horticulture, pharmaceutical
and cosmetic industry.

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Chapter 5
Molecular and Pathway Controls on Biogenic
Volatile Organic Compound Emissions

Ziru Li and Thomas D. Sharkey

Abstract Plants make a number of volatile organic compounds (BVOCs), many

of which are emitted in a light- and temperature-dependent manner. The vast
majority of these BVOCs are isoprenoids including isoprene, monoterpenes, and
sesquiterpenes. The total BVOC flux into the atmosphere is on the order of a
petagram (1015 g) and has multiple effects on atmospheric chemistry. Understanding
the biochemical and molecular regulation of BVOC emissions allows us to build
prediction models that better reflect the underlying physiological and biochemical
processes. In this chapter we review the enzymes and pathways involved in the
biosynthesis of various BVOCs that originate from plants, using isoprene as a
model. The biochemical and molecular control of BVOC emission in response to
short-term environment drivers such as temperature, light, CO2 , and O2 , and long-
term factors such as circadian, seasonal, and developmental effects are discussed.
An emerging theme in the regulation of isoprene emission is that the enzyme
isoprene synthase controls the basal emission rate in the long term, while the
responses of isoprene emission to short-term factors are regulated by levels of the
substrate (dimethylallyl diphosphate), which is in turn determined by upstream
enzymes. In addition, we propose a new hypothesis to explain the high-CO2
suppression of isoprene emission. At high CO2 concentrations, a high cytosolic
inorganic phosphate (Pi ) gradient needed to transport triose phosphates out of
the chloroplasts could work against the transport of phosphoenol pyruvate into
the chloroplasts. This altered partitioning of phosphoenol pyruvate would then
reduce the supply of pyruvate into the MEP pathway. Much work is still needed to
understand the CO2 response of BVOC emissions but we expect to see significant
progress in the near future.

Z. Li • T.D. Sharkey ()

Department of Biochemistry and Molecular Biology, Michigan State University,
East Lansing, MI 48824, USA
e-mail: [email protected]

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 119
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 5,
© Springer ScienceCBusiness Media Dordrecht 2013
120 Z. Li and T.D. Sharkey

5.1 Introduction

Understanding biological controls on biogenic volatile organic compound (BVOC)

emissions is one of the key topics in contemporary plant biology dealing with
plant abiotic and biotic stress resistance (Sharkey et al. 2008; Vickers et al.
2009a; Harrison et al. 2013). Furthermore, importance of BVOC emissions in
atmospheric reactivity and regional and larger scale Earth processes (Ashworth et al.
2013; Kulmala et al. 2013 in this volume) underscores the relevance of accurate
mechanistic description of BVOC emissions. Monoterpenes and isoprene make
up the largest group, in terms of mass, of BVOC. A mechanistic description of
biochemical and molecular regulation of monoterpene and isoprene emission allows
us to build bottom-up models that test our understanding of biochemical regulations
in different environments. These mechanistic models may be better at predicting
changes in BVOC emissions under very different scenarios such as predicting
isoprene emissions from the past (Possell et al. 2005; Schurgers et al. 2009) and
predicting the effects of climate change on isoprene emission (Arneth et al. 2007;
Young et al. 2009). A better understanding of the regulation of BVOC emissions
also opens up the possibility for engineering low-emitting species (Behnke et al.
2011; Rosenkranz and Schnitzler 2013 in this volume), which in the long term may
alleviate the impact of global climate change on BVOC emissions.
Many of the isoprenoids are synthesized in the chloroplast, and (along with
other secondary metabolic pathways) are downstream to metabolites in central
carbon metabolism, such as phosphoenol pyruvate (PEP) and glyceraldehyde
3-phosphate (GAP). Isoprenoid biosynthesis also shares a similar light response
with photosynthetic carbon assimilation suggesting a link of isoprenoid synthesis
to energetic cofactors produced in the light. Mechanistic models that have been
proposed to date generally link isoprene emission capacity or rate to some parameter
associated with photosynthesis (Niinemets et al. 1999; Martin et al. 2000; Zimmer
et al. 2000, 2003; Arneth et al. 2007). While this is a good step in the direction
of adding mechanistic understanding to empirical models, these models still are
limited by the lack of full mechanistic understanding of the regulation of key
biochemical pathways responsible for isoprenoid synthesis (Monson et al. 2012;
Grote et al. 2013; Monson 2013). Thus, there is opportunity for another big step
toward understanding properties of the enzymes of the major pathways supplying
substrates and the enzymes that convert these substrates to BVOC. This chapter
will cover recent advances in understanding biochemical and molecular regulations
of the enzymes involved in isoprenoid BVOC emissions from trees. Emission
mechanisms of other BVOCs including methanol, ethanol, acetic acid and green
leaf volatiles and modelling and engineering of BVOC emissions is covered in
several other chapters of the book (Ashworth et al. 2013; Grote et al. 2013; Guenther
2013; Harley 2013; Kreuzwieser and Rennenberg 2013; Rajabi Memari et al. 2013;
Monson 2013; Rosenkranz and Schnitzler 2013). Here we present an in depth look
at biochemical and molecular controls of isoprene emission and suggest the primary
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . . 121

areas for future experimental research to fill the gaps in knowledge and aid towards
development of fully mechanistic emission algorithms for plants stress studies and
for biosphere models.

5.2 What Volatile Isoprenoids Are Emitted?

Many compounds made by plants are volatile. In some cases, the volatility is
not a critical characteristic and in other cases the compound may be made non-
enzymatically (e.g., methane). Other BVOC may be made enzymatically but their
function may not be well known and the expected regulation unclear, for example
methanol and acetaldehyde. However, trees and other plants use enzymes to make
many compounds, especially isoprenoids, specifically because they are volatile.
Because of the roles isoprenoids play in response to abiotic stress (chapters by
Fineschi et al. 2013; Possell and Loreto 2013 in this volume) and biotic interactions
(Holopainen et al. 2013; Trowbridge and Stoy 2013), specific genes are expressed
to ensure the compounds are present when needed, but that carbon is not lost
unnecessarily when the volatile compounds are not needed. Some of the more
commonly emitted isoprenoids and their genetic and biochemical basis are covered
below.

5.2.1 Hemiterpenes

The most prominent hemiterpenes (C5 ) emitted from trees are isoprene (2-methyl-
1,3-butadiene) and methylbutenol (2-methyl-3-buten-2-ol, or MBO). Biogenic iso-
prene emission is the largest non-methane hydrocarbon flux into the atmosphere
and this large flux of volatile organic carbon has a profound effect on atmospheric
chemistry. For this reason, isoprene research has been active in the past and will be
a major focus in this review. Isoprene is mainly given off by broadleaved trees such
as aspens, oaks and eucalypts, as well as many legumes. MBO is only produced
in gymnosperms that make little isoprene. Both isoprene and MBO are produced
from dimethylallyl diphosphate (DMADP). DMADP with its isomer isopentenyl
diphosphate (IDP) are the functional isoprene units in plants and animals and the
building blocks of higher-order isoprenoids. DMADP and IDP in plants can be
synthesized through two pathways – a mevalonic acid (MVA) pathway that resides
exclusively in the cytosol and a methylerythritol phosphate pathway (MEP pathway,
also called the non-mevalonate pathway) in the plastids (Fig. 5.1). The relative sizes
of cytosolic and plastidic pools of DMADP and IDP can vary between species
and depend on the assay method used. Isoprene and MBO emitted from plants are
exclusively synthesized from the plastidic DMADP pool produced through the MEP
pathway.
122 Z. Li and T.D. Sharkey

Pyruvate GAP

DXS
CO2

DXP

NADPH + H+
DXR
NADP+

MEP

CTP
MCT
PPi

CDP-ME

MEP pathway
ATP
CMK
ADP

CDP-MEP

CMP MDS

MEcDP

Electrons
from ETC HDS

HMBDP

Electrons
from ETC HDR

IDP DMADP
IDI

IspS

Isoprene
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . . 123

5.2.2 Monoterpenes

The most abundant class of emitted hydrocarbons, in terms of the number of

compounds, are the monoterpenes. Among the well-known compounds are pinene,
limonene, and cineole (the scent of eucalypts). Each of these can exist as a variety
of isomers, for example, pinene can be ’ or “ (location of a double bond) and C or –
(stereochemistry of asymmetric carbon atoms) (Fig. 5.2). Monoterpenes are much
more varied than hemiterpenes. Monoterpenes can also be cyclic (pinene, limonene
etc.) or acyclic (myrcene, ocimene etc.). One of the more common oxygenated,
cyclic monoterpenes is cineole. Oxygenated acyclic monoterpenes include geraniol
(primary alcohol) and linalool (tertiary alcohol).
Monoterpenes also can be stored in structures such as resin ducts, trichomes
(hairs on the leaf surface), and glands in leaf tissue (especially in eucalypts). Other
monoterpenes are not stored but released as soon as they are made and some plants
will make both monoterpenes that are mostly stored and monoterpenes not stored.
Storage in specialized structures significantly affects the emission characteristics.
Emission of non-stored monoterpenes (and the never-stored hemiterpenes) is light-
dependent and the effect of temperature is on the metabolism producing the
compounds (see Harley 2013 for storage effects in “non-storing” emitters). For
stored monoterpenes, the effect of light will be secondary, operating through heating
effects, and physics of diffusion plays a dominant role in the rate of emission of these
monoterpenes.

5.2.3 Sesquiterpenes

In terms of the amount of volatile carbon released, sesquiterpenes likely represent

a much smaller source than isoprene or monoterpenes. This is in part because
sesquiterpenes are much less volatile, but the lack of observation of sesquiterpene
emission above tree canopies could also be influenced by their very short lifetime
in the atmosphere (Jardine et al. 2011). Sesquiterpene synthesis differs significantly
from isoprene and monoterpene synthesis. Sesquiterpenes are made by the cytosolic
mevalonic acid pathway, which produces the same precursors, IDP and DMADP
(Fig. 5.3). The next step is adding two IDP molecules to a DMADP to make farnesyl
diphosphate. Farnesyl diphosphate is then used by sesquiterpene synthase enzymes
to make sesquiterpenes.

J
Fig. 5.1 The MEP pathway. The MEP pathway provides substrates for all isoprenoids inside plas-
tids including the volatile isoprenoids isoprene and monoterpenes. The enzymes and metabolites
are defined in Table 5.1. ATP and CTP are regenerated by photophosphorylation and ferredoxin can
contribute electrons from the photosynthetic electron transport chain (ETC) without first passing
through NADPH. The requirement for ATP, CTP, and reducing power connects isoprene and
monoterpene synthesis to photosynthesis and is the basis for several models of emission rates
124 Z. Li and T.D. Sharkey

Fig. 5.2 Terpenes exist in

many similar forms. Pinenes
have two asymmetric carbon
atoms and the double bond
can be in either of two
locations, resulting in
different chiral (R/S or C/
or D/L) isomers. The acyclic
ocimenes have a double bond
that can be in either of two
places and the orientation
around another double bond
can vary, resulting in different
geometric (cis/trans or Z/E)
isomers

5.3 Molecular and Pathway Controls of Volatile

Isoprenoid Synthesis

5.3.1 The MEP Pathway

The existence of a mevalonic acid-independent pathway for isoprenoid synthesis

was not recognized until the early 1990s. Observed labelling patterns in certain
isoprenoids did not match predictions of a mevalonic acid pathway origin in
13
C-glucose- and 13 C-acetate-feeding experiments, leading to experiments that
resulted in the discovery of the MEP pathway. The MEP pathway was shown to
be the primary way that isoprenoids are produced in bacteria and in plastids of
plants (Lichtenthaler et al. 1997; Putra et al. 1998). This connects volatile emissions
of isoprene and monoterpenes with many other metabolic pathways including the
synthesis of nonvolatile compounds related to abiotic stress such as carotenoids and
abscisic acid.
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . . 125

Fig. 5.3 Isoprenoid biogenic volatile organic compounds are made either in the cytosol by
mevalonate (MVA) pathway (sesquiterpenes) or in the plastids by 2-C-methyl-D -erythritol
4-phosphate (MEP) pathway (hemi- and monoterpenes). In the cytosol, the mevalonic acid
(MVA) pathway converts acetyl-CoA to isopentenyl diphosphate (IDP). Some IDP is converted
to dimethylallyl diphosphate (DMADP) and one DMADP plus two IDP are combined to make the
sesquiterpene precursor farnesyl diphosphate. Most monoterpenes and isoprene are made inside
the chloroplast where the MEP pathway converts glyceraldehyde 3-phosphate and pyruvate into
IDP and DMADP for the production of isoprene (from DMADP) and monoterpenes (from the
precursor geranyl diphosphate)

An important step in understanding the new isoprenoid synthesis pathway was

the discovery that labeled 1-deoxy-D-xylulose was rearranged to a branched chain
(Arigoni et al. 1997). The first metabolite in the MEP pathway was shown to
be the 1-deoxy-D-xylulose 5-phosphate (DXP) originating from glyceraldehyde
3-phosphate and pyruvate (Rohmer et al. 1996). The first enzyme in the pathway
in plants was shown to be responsible for a lethal mutation known as cla1 (Mandel
et al. 1996). The second enzyme was discovered as the target of fosmidomycin
126 Z. Li and T.D. Sharkey

(Takahashi et al. 1998), an antibiotic that has proven to be useful in studying the
function of isoprene in trees.
Since then, great strides have been made toward elucidating the steps and
enzymes in this pathway, using a combination of reverse genetic and comparative
genomic approaches. The entire MEP pathway was elucidated by 2003 (Fig. 5.1
and Table 5.1). The first enzyme in the pathway, DXP synthase (DXS), forms
DXP from D-glyceraldehyde 3-phosphate and pyruvate. One CO2 molecule is
lost in the forward reaction towards DXP formation. DXP produced by DXS
is also a substrate in thiamine and pyridoxal synthesis. DXP reductoisomerase
(DXR) then catalyzes the formation of 2-C-methyl-D-erythritol phosphate (MEP),
the first committed metabolite that also gives this pathway its name. The next
enzyme, MEP cytidyltransferase (MCT) transfers a cytidyl moiety from CTP to
form 4-(cytidine 50 -diphospho)-2-C-methyl-D-erythritol (CDPME) with the release
of a pyrophosphate. This compound is phosphorylated by CDPME kinase (CMK) to
produce CDPME 2-phosphate (CDPMEP), which is then cyclized to 2-C-methyl-D-
erythritol 2,4-cyclodiphosphate (MEcDP) by MEcDP synthase (MDS) with the loss
of the cytidyl group. Finally, 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBDP)
is produced by HMBDP synthase in the penultimate step in the pathway, and is then
converted to DMADP and IDP by HMBDP reductase (HDR) in an approximately
1:5 ratio (Rohdich et al. 2002). Under steady-state conditions the equilibrium
ratio between DMADP and IDP is approximately 2:1, and the isomerization is
accelerated in vivo by an IDP isomerase (IDI) that is present in both the cytosol
and the chloroplast. Modelling of this pathway requires information on the kinetic
constants for all of the enzymes but in many cases these are only known from
bacterial enzymes (Table 5.2).

5.3.2 Isoprenoid Synthases

Most hemiterpenes and monoterpenes emitted to the atmosphere are made by

proteins coded by genes in the terpene synthase (Tps) family. The Tps family can
be traced back evolutionarily to a gene in the moss Physcomitrella patens (Fig. 5.4)
(Tholl 2006; Chen et al. 2011). This gene codes for kaurene synthase, an important
step in the synthesis of the plant hormone gibberellin. The Tps genes have evolved
to provide many important terpenoids in most lands plants (Trapp and Croteau
2001). Two of the main sections of this family are responsible for the majority of
hydrocarbons emitted by trees (Bohlmann et al. 1998; Rajabi Memari et al. 2013;
Rosenkranz and Schnitzler 2013).

5.3.2.1 Isoprene Synthase

Isoprene synthesis from DMADP is catalyzed by the enzyme isoprene synthase

(IspS) (the abbreviation IspS will be used italicized when describing the gene and
Table 5.1 Nomenclature of enzymes in the MEP pathway with corresponding genes in the bacterium Escherichia coli and in the annual vascular plant
Arabidopsis thaliana
Other
Step Enzyme name EC number Abbreviation abbreviations E. coli gene A. thaliana gene
1 1-Deoxy-D-xylulose 5-phosphate 2.2.1.7 DXS dxs DXS (At4g15560)
synthase
2 1-Deoxy-D-xylulose 5-phosphate 1.1.1.267 DXR ispC (yaeM, dxr) DXR (At5g62790)
reductoisomerase
3 2-C-Methyl-D-erythritol 4-phosphate 2.7.7.60 MCT MECT, CMS ispD (ygbP) MCT (At2g02500)
cytidylyltransferase
4 4-(Cytidine 50 -diphospho)-2-C-methyl- 2.7.1.148 CMK CMEK ispE (ychB) CMK (At2g26930)
D-erythritol kinase
5 2-C-Methyl-D-erythritol 4.6.1.12 MDS MECPS, MECS, MCS ispF (ygbB) MDS (At1g63970)
2,4-cyclodiphosphate synthase
6 4-Hydroxy-3-methylbut-2-enyl 1.17.7.1 HDS ispG (gcpE) HDS (At5g60600)
diphosphate synthase
7 4-Hydroxy-3-methylbut-2-enyl 1.17.1.2 HDR IDS ispH (lytB) HDR (At4g34350)
diphosphate reductase
Modified from Phillips et al. (2008)
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . .
127
128 Z. Li and T.D. Sharkey

Table 5.2 Available estimates of kinetic parameters of MEP pathway enzymes

Enzymes Substrate Km (mM) kcat (s1 ) References
DXS GAP 0.12* 14* Kuzuyama et al. (2000), Hahn et al.
Pyruvate 0.096* (2001), Bailey et al. (2002),
Eubanks and Poulter (2003), Lee
et al. (2007), and Brammer and
Meyers (2009)
DXR DXP 0.14 4.4 Engprasert et al. (2005), Rohdich et al.
NADPH 0.056 (2006), Jawaid et al. (2009), and
Takenoya et al. (2010)
MCT MEP 0.50 26 Rohdich et al. (2000)
CTP 0.11
CMK CDPME 0.14* – Bernal et al. (2005) and Sgraja et al.
ATP 0.32* (2008)
MDS CDPMEP 0.48 2.5 Geist et al. (2010)
HDSa MEcDP 0.56 0.4* Kollas et al. (2002), Seemann et al.
(2005, 2006), and Zepeck et al.
(2005)
HDRa HMBDP 0.31* 3.7 Altincicek et al. (2002); Gräwert et al.
(2004)
IDI IDP 0.0057 0.69* Spurgeon et al. (1984), Jones et al.
(1985), Dogbo and Camara (1987),
and Lützow and Beyer (1988)
IspS DMADP 2.5 1.8 Silver and Fall (1995), Wildermuth and
Fall (1996), Schnitzler et al. (2005),
Wiberley et al. (2008) and Rasulov
et al. (2009a, b)
If kinetic data from a plant enzyme is available, then only data for the plant enzymes are used,
otherwise values are derived from bacterial enzymes and are denoted by an asterisk. The median
values of reported estimates for each enzyme are listed
a
The HDS enzyme in Arabidopsis can obtain electrons directly from photosynthesis, possibly via
ferredoxin (Seemann et al. 2006). HDR displays activity in presence of ferredoxin/ferredoxin-
NADPC /NADPH system, but its electron source in plants is less clear (Rohdich et al. 2002). No
kinetic data has been reported yet for the second substrate (the electron donor) for either of the two
enzymes

not italicized when referring to the protein), a close relative to other monoterpene
and diterpene synthases and a member of the Tps-b family (Miller et al. 2001).
IspS in major emitting species has a plastid-targeting sequence and is localized
to the chloroplasts. Due to its high volatility, isoprene emitted from plants does
not build to substantial amounts within the leaf, but instead passes through two
membrane systems (the chloroplast membranes and plasma membrane) and is
released into the atmosphere. High Km values have been reported for isoprene
synthase (0.5–8 mM) suggesting that substrate concentration can play an important
role in regulation of isoprene emission. Reported values for kcat for isoprene
synthase range from 0.03 to 0.26 s1 (Gray et al. 2011). The isoprene synthases
that have been sequenced up to now belong to a single clade within the Tps-b group
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . . 129

Fig. 5.4 Phylogenetic tree of a subset of genes making enzymes important for hydrocarbon
emissions from trees, terpene synthases (Tps). The amino acid sequences of these genes were
obtained from NCBI. The genes were selected to show the relationship of the Tps genes between
gymnosperms (Tps-d) and angiosperms (Tps-g and Tps-b). Abbreviations for gene products are
Kau, kaurene; Lim, limonene; MBO, methyl butenol; Pin, pinene; Lin, linalool; Oci, ocimene;
Ner, nerolidol; Isp, isoprene; Cin, cineole. The brown box shows the ocimene/isoprene synthase
clade, blue indicates gymnosperms, green is Tps-g genes and red Tps-b genes of angiosperms. The
tree was constructed based on a Bayesian analysis (Mr. Bayes 3.2) as described by Sharkey et al.
(2012)
130 Z. Li and T.D. Sharkey

of terpene synthases (Fig. 5.4 and Sharkey et al. 2013). In addition to isoprene
synthases, this clade has genes that code for E-“-ocimene synthases but no other
monoterpenes. This clade occurs only in the rosid group of angiosperms. It is
likely that emission from non-rosid angiosperms, for example palm and bamboo,
results from a different gene type and that this other isoprene synthase arose
independently. It is now believed that IspS evolved ca. 100 million years ago in
multiple lineages and is a trait that has been gained and lost multiple times, similarly
to the evolutionary history of C4 photosynthesis (Monson et al. 2013; Sharkey et al.
2013).

5.3.2.2 Methylbutenol Synthase

Enzymatic production of MBO from DMADP was first demonstrated in pine

needles by Fisher et al. (2000). The MBO synthase cloned from Pinus sabiniana
produces multiple products, primarily making MBO, but also producing isoprene
in trace amounts (Fisher et al. 2000; Gray et al. 2011). This propensity to make
MBO is enhanced in vivo by the KC dependence of this enzyme, where it has
been shown that MBO production increases and isoprene production decreases with
increasing KC concentrations. At physiological concentrations of KC in leaves,
MBO synthase produces very little or no isoprene which can explain why no
isoprene emission is observed from pine trees. The Km of MBO synthase (10–
20 mM) is high, although comparable to those of angiosperm isoprene synthases,
while kcat is comparable to monoterpene and sesquiterpene synthases. The MBO
synthase evolved independently from angiosperm isoprene synthases and falls into
the Tps-d1 group.

5.3.2.3 Monoterpene Synthases

The monoterpenes are made by terpene synthases from geranyl diphosphate [with a
few exceptions, for example phellandrene made in tomato trichomes from neryl
diphosphate, an isomer of geranyl diphosphate (Schilmiller et al. 2009)]. Like
isoprene, the precursor for monoterpenes is made by the MEP pathway inside
chloroplasts. The IDP and DMADP made by the MEP pathway are combined
head to tail by geranyl diphosphate synthase resulting in a new allylic diphosphate
molecule, geranyl diphosphate.
The gymnosperms have Tps-d genes and the Tps-d1 sub-group has most of the
genes responsible for volatiles made by gymnosperm trees (Martin et al. 2004;
Rajabi Memari et al. 2013; Rosenkranz and Schnitzler 2013). In the angiosperms
a different group of genes are responsible for hemi- and monoterpene synthase
enzymes. Tps-g genes (denoted by green in Fig. 5.4) are related to Tps-b but are
more rare [with new genomes being analysed the number of known Tps-g genes is
increasing (Martin et al. 2010)]. Tps-g proteins always make acyclic monoterpenes
such as ocimene and linalool. The ocimene synthases are often relatively unspecific,
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . . 131

making especially myrcene in addition to ocimene. Within the Tps-b genes is a

clade made up of ocimene synthases and isoprene synthases (denoted by brown in
Fig. 5.4). The ocimene synthases of the ocimene/isoprene synthase clade are specific
for ocimene synthase and do not make other monoterpenes. Ocimene synthases in
other sections of the Tps-b often make ocimene and myrcene and sometimes other
monoterpenes as well.
Usually enzymes are stereospecific, but Tps enzymes frequently make a variety
of products including different isomers. Isomers can be defined by location of
double bonds (typically labeled ’ or “) and can also be related to the orientation
around double bonds (E, or cis versus Z or trans) or the orientation at asymmetric
carbon atoms (chirality). Four isomers of pinene (bicyclic) and ocimene (acyclic)
are shown in Fig. 5.2. The lack of strict isomeric specificity in many Tps enzymes
is unusual as is the formation of multiple products from one enzyme.
It is believed that the lack of specificity in Tps enzymes results from the reaction
mechanism. For hemi- and monoterpenes an allylic diphosphate precursor first loses
its diphosphate to make a carbocation. The presence of a positive charge on a
molecule with double bonds makes a highly unstable reaction intermediate that
is quenched by water to make oxygenated terpenes or abstraction of a proton to
make non-oxygenated terpenes. The specific compound formed and its isomeric
conformation will depend on which proton is abstracted and the conformation of the
active site. It has become possible to predict how to interconvert product specificity
by changing very few amino acids (Katoh et al. 2004; Hyatt and Croteau 2005;
Kampranis et al. 2007). Because product specificity is so variable, terpene synthases
tend to group by phylogenetic considerations more than by the products they make.
For example, the two Arabidopsis thaliana genes in Fig. 5.4 (and the other five Tps-
b genes of Arabidopsis, data not shown) group together while monoterpene synthase
genes of spices (bottom three species in Fig. 5.4) form a single group (even when
over 70 Tps-b genes are included in the analysis, Sharkey et al. 2013). The products
shown in Fig. 5.4 vary in location on this phylogenetic tree, for example limonene
is found at the top and bottom. On the other hand, enzymes with very different
products from closely related species tend to be closely grouped.

5.3.2.4 Sesquiterpene Synthases

Sesquiterpene synthases are the enzymes forming specific sesquiterpenes from

farnesyl diphosphate. They are typically Tps-a in the case of angiosperms and
typically Tps-d2 and d3 in the case of gymnosperms. The division between chloro-
plast (C5, C10, C20, C40) and cytosolic (C15, C30) terpene synthase activities is
reasonably strict but it is not clear why this should be. Like monoterpene synthases,
sesquiterpene synthase product specificity can be manipulated by changing a limited
number of amino acid residues (Yoshikuni et al. 2006; O’Maille et al. 2008).
To ensure that the Tps enzymes are in the right compartment a transit sequence
of about 50 amino acids is added to the hemi- and monoterpene gene sequences
that targets them to the chloroplast and is cleaved once the gene product is inside
132 Z. Li and T.D. Sharkey

the chloroplast. Almost all Tps-b genes have a transit sequence but Tps03 of Ara-
bidopsis thaliana does not (Huang et al. 2010). Tps03 is nearly identical to Tps02,
which does have a transit sequence. Both enzymes can make ocimene or farnesene
depending on the substrate provided. It appears that Arabidopsis uses the presence or
absence of a transit sequence to cause one enzyme to make ocimene (in the chloro-
plast) while the other makes farnesene (in the cytosol). The advantage of separating
sesquiterpene synthesis from hemi- and monoterpene synthesis is not clear.

5.4 Environmental Regulation of Monoterpene

and Isoprene Emissions

Monoterpenes and sesquiterpenes are often stored in storage bodies (e.g., resin
ducts or trichomes) and can accumulate to significant levels within plant tissues.
However, relatively little is known about the biochemical and genetic controls of
the accumulation of compounds in these structures and release from these structures
is more a matter of physics, and less biology. There are some projects designed to
elucidate how trichome biochemistry, for example, is controlled and it has been
observed that in some cases, gene expression is very tightly controlled so that it
occurs only in the tips of trichomes (Schilmiller et al. 2008, 2010). Compounds that
are soluble can accumulate inside leaf tissue which can cause effects of stomatal
opening on emission rates in addition to the effects of biochemistry (Copolovici
and Niinemets 2005). On the other hand, isoprene has a relatively high Henry’s law
constant (a measure of the partitioning between air and water) and so is emitted
essentially as soon as it is made by isoprene synthase. Initially the lack of stomatal
effects on isoprene emission rate or kinetics was interpreted to mean that isoprene
diffuses through the leaf epidermis instead of through stomata (Monson and Fall
1989). However, it was subsequently shown that changes in stomatal conductance
caused compensating changes in isoprene concentration inside the leaf making
isoprene emission independent of stomata (Sharkey 1991; Fall and Monson 1992).
The rate of emission of isoprene is therefore much more dependent on the regu-
lation of enzymes involved and more responsive to rapid changes in environmental
variables. In particular, isoprene emission rates are characterized by rapid fluctua-
tions in natural environments, presumably driven by changes in leaf temperature due
to rapid conductive heat exchange from the surrounding air currents. Both isoprene
and MBO emissions are characterized by strong light and temperature dependencies
(Monson and Fall 1989; Harley et al. 1998; Gray et al. 2005). Isoprene scales
positively with light, and increases with increasing temperature until 40–45 ı C,
at which temperature emission rates fall sharply. Isoprene emission in many species
decreases with increasing CO2 concentrations, and the cause of this high- CO2
suppression effect is unclear. The following sections will discuss and summarize
present knowledge regarding regulation of BVOC emissions from a biochemical and
molecular biology perspective, using studies of isoprene emission as an example.
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . . 133

5.4.1 Short-Term Effects

Isoprene emission (and emission of some monoterpenes) responds very quickly

to changes in the environment, for example changes in light, temperature, and
CO2 . Short-term effects are generally interpreted in terms of changes in metabolite
pool sizes and availability of energetic intermediates such as ATP and NADPH.
Over time, the biochemical control of the short-term temperature response has
become more clear. The short-term light response has been assumed to be related to
photosynthetic electron transport. Additional short-term effects of CO2 and O2 are
now recognized but the underlying mechanisms are still being actively studied.

5.4.1.1 Temperature

Isoprene emission varies strongly with leaf temperature (Sanadze and Kalandaze
1966). The response to leaf temperature is extremely rapid (Singsaas and Sharkey
1998). Unlike photosynthesis which has a maximum at 25–30 ı C, isoprene emission
exhibits a strong temperature dependence up to 40–45 ı C, a temperature which
is often deleterious to photosynthesis (Sharkey 2005). The reported temperature
maxima for isoprene emission differ by as much as by 8 ı C, and this is to a
large extent due to differences in the measurement methodologies (Monson et al.
1992; Singsaas and Sharkey 2000). Isoprene emission at temperatures above 40 ı C
is not sustainable for an extended period of time (usually less than 20 min), due
to a shortage of substrates (Li et al. 2011). Therefore, depending on how fast
leaf temperature was elevated, and whether the same leaf or different leaves were
used for different temperature points, the temperature responses will vary. The
relatively large pools of MEP pathway intermediates support very high rates of
isoprene synthesis for short periods during heat flecks; when leaves return to lower
temperature, these pools can refill to be ready for the next heat fleck (Singsaas and
Sharkey 1998).
It was known for a long time that IspS activity increases greatly with temperature
and the link between emission rate and isoprene synthase activity has been
postulated from very early on (Monson et al. 1992). It was also noticed that the
optimum for IspS is higher than that of isoprene emission, and the possibility of a
substrate-side limitation was raised (Lehning et al. 1999). Rasulov et al. (2009a)
developed a method for estimating DMADP levels in vivo by measuring post-
illumination isoprene emission. In addition, we recently presented evidence that
a post-illumination isoprene burst is a good approximation for other intermediate
metabolites in the MEP pathway (Li and Sharkey 2013). Using these techniques
it has been shown that the temperature at which DMADP accumulates the most
is 35 ı C, and this is the same for intermediate metabolites in the MEP pathway
(Rasulov et al. 2010; Li et al. 2011; Rasulov et al. 2011). The optimum temperature
for IspS on the other hand is 50 ı C with an activation energy of approximately
40–50 kJ mol1 (Monson et al. 1992; Lehning et al. 1999; Rasulov et al. 2010; Li
et al. 2011).
134 Z. Li and T.D. Sharkey

The increase in substrate availability combined with the increased IspS activity
as temperature is increased up to 35 ı C results in a very high temperature sensitivity
of isoprene emission, exceeding the temperature sensitivity of isoprene synthase.
Between 35 and 40 ı C the substrate concentration declines, but IspS activity
increases, giving an overall higher isoprene emission rate. Above 40–45 ı C, the
decline in substrate outweighs the stimulatory effect of temperature on IspS result-
ing in reduced isoprene emission as temperature goes above 40–45 ı C. Empirical
models fit equations thought to predict single enzyme responses to temperature
(Guenther et al. 1993) but, while these work well, they do not have a mechanistic
basis given that substrate concentration changes and effects of temperature on kcat
contribute, in varying proportions, to the overall temperature response of isoprene
emission. It is now generally accepted that the response of isoprene emission to
temperature results from the thermodynamic properties of the enzymes involved,
and the control is shared between the enzyme IspS, and the MEP pathway enzymes
that determine DMADP levels. While enzymes in the MEP pathway generally
have a temperature optimum that is somewhat above the ambient temperature [e.g.,
37 ı C for DXR (Rohdich et al. 2006)], the temperature optimum for IspS is even
higher; such that, isoprene emission is characterized by a marked temperature
response (up to 40–45 ı C), while synthesis of other downstream housekeeping
isoprenoids, e.g., carotenoids and quinones, are presumably much less so. It might
be interesting to speculate why this has evolved to be the case. Isoprene may play a
role in protecting plants against moderate heat stress on hot summer days when leaf
temperatures frequently reach but usually do not go much beyond 40 ı C (Sharkey
et al. 2008).

5.4.1.2 Light

Historically, two hypotheses had been put forward to explain the light response
of isoprene emission: (1) changes in DMADP levels (Loreto and Sharkey 1993;
Rosenstiel et al. 2002; Rasulov et al. 2009b) and (2) changes in IspS activation
state (Wildermuth and Fall 1996; Fall and Wildermuth 1998; Sasaki et al. 2005).
While transcription of IspS is light-dependent (Sasaki et al. 2005) and IspS appears
to be under circadian regulation (discussed later), transcription and translation of
genes typically take place on a longer timescale and cannot explain the instanta-
neous responses to light levels. Measurement of DMADP content by non-aqueous
fractionation (Rosenstiel et al. 2002), post-illumination isoprene emission (Rasulov
et al. 2009b) and mass spectrometry (Li and Sharkey 2013) showed that DMADP
content varies, while calculated isoprene synthase activity stays roughly constant
with varying light intensities. These pieces of evidence suggest substrate-level
control of isoprene emission. The upstream enzymes in central carbon metabolism
and the MEP pathway that determine DMADP levels both require a significant
amount of ATP and NADPH, presumably provided by the light reactions of
photosynthesis.
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . . 135

Fig. 5.5 Illustration of kinetic changes of isoprene emission from a leaf of hybrid poplar (Populus
tremula) following light–dark transients and changes in ambient gas composition (Modified from
Li and Sharkey 2013). When the light is turned off, isoprene emission from leaves falls rapidly
but then shows a short post-illumination burst. When reilluminated, the leaf will resume making
isoprene as much as before. When a leaf is subjected to O2 - and CO2 -free air (i.e., 100 % N2 )
isoprene emission is rapidly inhibited but no subsequent burst is observed. Resupplying O2 and
CO2 allows isoprene emission to go to very high rates but it does not fully recover

The question then becomes: which are the upstream steps that control light-
dependent changes in DMADP? An important clue was gained from studies of
isoprene emission during light-dark transients. When light is turned off on an
emitting leaf, isoprene emission rapidly decreases to almost zero within 8–10 min
(phase I) (Fig. 5.5). Emission level usually then starts to increase again in the dark,
peaks at 20–25 min before dropping off again to zero in approximately 45 min
(phase II). Timing of the so-called “post-illumination isoprene burst” is temperature-
dependent, and the burst occurs sooner at higher temperatures. Measurement of
MEP pathway metabolites during this period shows intermediate metabolites in
the MEP pathway, primarily MEcDP, stays at approximately the same level during
phase I when isoprene emission declined by >90 %. Later, MEcDP was converted
to isoprene, forming the post-illumination burst. Therefore, the decline of isoprene
emission during phase I can be explained by a rapid depletion of reducing power,
inhibiting HDS (albeit incompletely). During photosynthesis, NADPH turns over
faster than any other photosynthetic metabolite and can have a half-life of just 10 ms,
compared to ATP with a half-life of 280 ms (Arrivault et al. 2009).
The inhibition of HDS is then reversed in the first part of phase II leading to
an increase in emission levels. NADPH could be regenerated through the pentose
phosphate pathway or plastidic glycolysis; alternatively, the switch of HDS from
using ferredoxin to NADPH as a reducing power source may take time. What
136 Z. Li and T.D. Sharkey

causes the eventual decline in isoprene emission (later part of phase II) is less clear.
NADPH presumably has already been regenerated as seen in the post-illumination
isoprene burst, and it is also needed for anabolic cellular processes in the dark. At
this time, all of the MEP pathway metabolites dropped to minimal levels (Li and
Sharkey 2013). This suggests steps in the central metabolism upstream of DXS
have been turned off, cutting off the carbon supply to the MEP pathway. GAP
is likely to be the limiting substrate as GAP levels were quickly reduced upon
darkness while levels of 3-phosphoglyceric acid (3-PGA), from which pyruvate is
made, accumulates initially in darkness (Sharkey et al., 1986; Loreto and Sharkey
1993). We suggest that the darkness-induced reduction in GAP levels results from
the loss of redox power to convert PGA to GAP rather than a simple consequence of
reduced carbon assimilation, since substantial isoprene emission can be seen under
photorespiratory conditions (e.g., CO2 -free air) where the carbon balance is more
negative than the carbon balance in darkness. The tight physiological control in
darkness decreased isoprene emission to essentially zero but when light is turned
back on emission capacity is fully reversible. This is in sharp contrast to isoprene
emission in N2 (i.e., no O2 and no CO2 ), where the disruption of redox balance is
non-physiological; despite a strong inhibition at HDS, a trace amount of isoprene is
still emitted in N2 , and isoprene emission capacity is irreversibly damaged after the
treatment (Fig. 5.5 and Li and Sharkey 2013).

5.4.1.3 CO2 and O2

Starting from CO2 -free air, isoprene emission often increases with increasing CO2
concentrations until 50 mol mol1 CO2 , or approximately the CO2 compen-
sation point of photosynthesis, where emission levels off and then sometimes
decreases with increasing CO2 concentration. The short-term decrease in isoprene
emission with increasing CO2 has been seen often but not universally. The CO2
response is temperature-dependent, and the high CO2 suppression effect goes away
at higher temperatures (Rasulov et al. 2010). This interaction between temperature
and CO2 effects could be important for modelling considerations but has so far
gained little recognition.
The suppression of isoprene at high CO2 concentration is perplexing. Judging
from the CO2 response of photosynthesis, we would predict isoprene emission
to increase, not decrease, with increasing CO2 . Glyceraldehyde 3-phosphate (an
end-product of photosynthesis) is one of the two substrates for the MEP pathway,
and 13 CO2 -labelling studies have shown that under standard conditions a large
proportion of isoprene emission comes from recent photosynthates (Delwiche and
Sharkey 1993; Karl et al. 2002; Loreto et al. 2004). Interestingly, in CO2 -free air, a
substantial amount of isoprene is emitted, at a rate that is comparable to emission
at ambient CO2 levels. Isoprene emission from leaves in CO2 -free air decreases
slowly over time, but still does not reach zero after >10 h (Li, Z. and Sharkey, T.D.
unpublished data). Carbon required for isoprene synthesis could obviously come
from an alternative source (e.g., transitory starch).
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . . 137

Measurements of DMADP levels by non-aqueous fractionation showed that

the CO2 response of isoprene emission is regulated by substrate levels. Based on
this experiment, Rosenstiel et al. (2003) proposed that CO2 response of isoprene
emission reflects a competition for PEP between PEP carboxylase in the cytosol and
import into the chloroplast through the Pi /PEP transporter (PPT) for conversion to
pyruvate. An alternative hypothesis is that energetic cofactors required for the MEP
pathway, such as ATP and NADPH, are affected at high CO2 conditions (Rasulov
et al. 2009b). As CO2 concentrations increase, photosynthesis switches from being
limited by Rubisco to being limited by linear electron transport that generates ATP
and NADPH (Farquhar et al. 1980). At higher CO2 concentrations, photosynthesis
can be also feedback-limited by inorganic phosphate levels which is determined
by the speed of triose phosphate synthesis relative to its consumption by starch
and sucrose synthesis (Sharkey 1985). This decrease in phosphate levels reduces
ATP synthesis (Sharkey and Vanderveer 1989; Kiirats et al. 2009). However, it is
important to note that cellular phosphate levels could have multiple regulatory roles.
Here we propose yet another explanation for the CO2 inhibition of isoprene
synthesis. We suggest that reduced plastidic phosphate levels at high CO2 con-
centrations affect the equilibrium across the Pi /PEP antiporter on the chloroplast
membrane, and reduce PEP concentration in the chloroplasts without a reduction
in PEP levels in the cytosol. A Pi gradient from outside to inside the chloroplast
is required to move triose phosphate out of chloroplasts for sucrose synthesis and
for PGA export for PEP synthesis (Fig. 5.6). This would work against import of
PEP and so could limit the supply of pyruvate for the MEP pathway. Sucrose
synthesis has a very high temperature sensitivity and so the Pi gradient working
against PEP import into the chloroplast might decline at high temperature (Sage
and Sharkey 1987; Stitt and Grosse 1988). This would explain why CO2 inhibition
of isoprene emission often disappears at moderate to high temperature. One way
to distinguish PEP carboxylase competition from reduced PEP import because of
unfavorable PEP distribution within the cell is to measure PEP levels within the
leaf as a function of CO2 . We know of no such measurements in isoprene-emitting
species, but there is a report of PEP levels in Arabidopsis leaves as a function of
CO2 (Arrivault et al. 2009). These investigators found a strong increase in PEP as
CO2 was increased, consistent with the PEP distribution hypothesis and inconsistent
with the PEP carboxylase competition hypothesis.
In addition to PEP import, a sodium-dependent pyruvate transporter has been
reported (Furumoto et al. 2011). It is not clear whether this is active during the day or
how changing CO2 might change the distribution of pyruvate across the chloroplast
envelope. Additional work, including determining partitioning of PEP and pyruvate
between the cytosol and chloroplast in isoprene-emitting species will help resolve
the biochemical basis of the CO2 suppression of isoprene emission.
Under low O2 conditions, isoprene emission typically increases. Lower levels
of photorespiration could lead to an increased availability of energetic cofactors,
increasing the capacity for isoprene synthesis. In the absence of both CO2 and
O2 (N2 conditions, no O2 and no CO2 ), isoprene emission is quickly abolished.
Metabolic profiling of the MEP pathway showed that HMBDP synthase (HDS, step
138 Z. Li and T.D. Sharkey

Fig. 5.6 Supply of carbon for isoprene synthesis from photosynthesis. The primary product of
photosynthesis, glyceraldehyde 3-phosphate, can be used directly from the Calvin-Benson cycle,
but chloroplasts generally do not have phosphoglucomutase to convert 3-phosphoglyceric acid
(3-PGA) to 2-PGA. This may help metabolism in the chloroplasts to go in the direction of
sugar synthesis. Glyceraldehyde 3-phosphate and 3-PGA can be exported from the chloroplast
by exchange for phosphate on the triose phosphate/phosphate antiporter. PEP can be taken up by
a PEP/phosphate transporter. However, the phosphate gradient that must be high in the cytosol to
favor glyceraldehyde 3-phosphate export, will make PEP import difficult

6 of the MEP pathway) is strongly inhibited under this condition causing a 30-
fold increase in substrate levels. Switching off both CO2 and O2 would inhibit both
the carboxylation and oxygenation reactions of Rubisco, in essence turning off the
Calvin-Benson cycle, which would disrupt cellular redox balance. The plant HDS
is an oxygen-sensitive enzyme (Seemann et al. 2002) and the altered cellular redox
potential may lead to increased enzyme turnover (Rivasseau et al. 2009).
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . . 139

5.4.2 Long-Term Effects

In contrast to the short-term responses dominated by biochemical regulations,

longer-term effects are increasingly recognized and these are generally related to
molecular controls such as changes in gene expression and even the presence or
absence of specific genes. For example, species differences in whether or not a plant
emits isoprene may result primarily from the presence or absence of a functional
isoprene synthase. Transformation of Arabidopsis (Sharkey et al. 2005; Sasaki
et al. 2007) or tobacco (Vickers et al. 2009b) with an isoprene synthase gene can
cause a species that normally does not emit isoprene to begin emitting isoprene.
The emission rate measured or corrected to 30 ı C and photon flux density of
1,000 mol m2 s1 (to control for short-term effects) varies over the course of
a day, over the course of a season, in response to weather, and by leaf location
within a canopy. Overall, we note that it is difficult to deconvolute ontogenetic and
environmental effects on isoprene emission and the observed responses may often
actually reflect combined effects of environmental variations and leaf age.

5.4.2.1 Circadian Effects on Isoprene Emission Capacity

There have been several reports of circadian changes in isoprene emission capacity
(Funk et al. 2003; Wilkinson et al. 2006; Loivamäki et al. 2007; Wiberley et al.
2008, 2009). When measurements are made under ambient conditions, the circadian
changes in emission can result from light or temperature changes. However, as early
as in 1986, it was recognized that some of the circadian change in isoprene emission
was beyond what could be accounted for by changes in light (Ohta 1986). In some
plants, the circadian effect can be absent or modest (Lehning et al. 1999), but when
measured at high temperature or photon flux density, the effect is greater (Wilkinson
et al. 2006). When measured under constant light, isoprene emission capacity in
poplar leaves exhibits an ultradian cycle with a 12 h period (Wiberley et al. 2009).
Trees grown at 30 ı C show a more pronounced circadian effect than trees grown
at 20 ı C (Wiberley et al. 2008). The amount of mRNA for IspS also varies over
the course of the day but these variations are not reflected in measurable protein
amounts (Wiberley et al. 2009). Several enzymes of the MEP pathway, especially
DXS and HDR also show very large circadian patterns in mRNA accumulation
(Wiberley et al. 2009), but again there is no evidence for significant changes in
protein amount. The half-life of IspS protein was estimated to be 5.3 days for
trees grown at 20 ı C and 3.4 days for trees grown at 30 ı C. Little is known
about the relative availability of substrate through the day, so the importance of
the MEP pathway versus isoprene synthase regulation for circadian effects is not
yet known.
140 Z. Li and T.D. Sharkey

5.4.2.2 Seasonality

Seasonal changes in isoprene emission capacity have been reported many times
(Monson et al. 1994; Guenther 1997; Schnitzler et al. 1997; Goldstein et al. 1998;
Fuentes and Wang 1999; Fuentes et al. 1999; Zhang et al. 2000; Pegoraro et al.
2007). There is some evidence for changes in mRNA levels for IspS, but with
a strong effect of temperature interacting with seasonal effects (Mayrhofer et al.
2005).

5.4.2.3 Weather

The seasonal effects reflect two other effects, a weather effect and a developmental
effect. The weather effect refers to the fact that a period of several warm days
results in higher isoprene emission capacity than a period of several cool days.
This effect has been seen many times (Sharkey et al. 1999; Geron et al. 2000;
Pétron et al. 2001). This has also been seen for methylbutenol (Gray et al. 2006)
even though the gene for methylbutenol synthase has a very different evolutionary
history from known isoprene synthases (Gray et al. 2011). The time period over
which temperature effects affect isoprene emission capacity has been found to be
anywhere from 6 h to 15 days. This effect has been studied in trees and mosses
(Hanson and Sharkey 2001a, b; Wiberley et al. 2008). There is evidence for changes
in the amount of isoprene synthase enzyme that can account for some of the effect
of weather on isoprene emission capacity (Wiberley et al. 2008).

5.4.2.4 Developmental Effects

Isoprene emission capacity develops more slowly during leaf development than does
the capacity for photosynthesis and this effect is temperature-dependent (Grinspoon
et al. 1991; Kuzma and Fall 1993; Sharkey and Loreto 1993; Harley et al. 1994).
The extractable activity of isoprene synthase can account for this effect (Kuzma and
Fall 1993). More recently it was shown that the temperature-dependent delay in the
onset of isoprene emission capacity was regulated by expression of the isoprene
synthase gene (Wiberley et al. 2005; Sharkey et al. 2008). It is also likely that as
leaves senesce, isoprene synthase is degraded, resulting in time-dependent reduction
of isoprene emission rates (Sun et al. 2012).
Seasonal effects are treated as a separate phenomenon but they may reflect a
combination of weather and developmental effects (Grote et al. 2013 for further
discussion of difficulties in describing seasonality in models). In any case, empirical
models include algorithms for approximating the changes in isoprene emission
capacity through the season and this has improved model performance.
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . . 141

5.4.2.5 Canopy Location

Leaves at the top of a canopy will be hotter during the day, colder at night, and
exposed to much more light than leaves in the middle of a canopy. It has been found
that leaves at the top of a canopy emit significantly more isoprene than leaves at the
bottom of a canopy (Sharkey et al. 1996; Niinemets et al. 2010). The analysis of
Niinemets et al. (2010) showed that isoprene emission capacity was well-correlated
with light availability, but it is also possible that temperature differences of leaves at
different locations in the canopy contribute to the differences in isoprene emission
capacity at different locations in a canopy.

5.4.2.6 Ozone and CO2

Trees grown in high [CO2 ] or high ozone reduced isoprene emission capacity
(Calfapietra et al. 2007). Elevated [CO2 ] caused a very slight reduction in message
level for IspS and slightly more reduction in protein level, though neither was
statistically significant. In elevated ozone both message level and protein level were
significantly reduced and the presence or absence of elevated [CO2 ] had no further
effect.
The effect of elevated [CO2 ] on the long-term capacity for isoprene emission
has sometimes been interpreted in the same way as the short-term effects but
Sun et al. (2012b) challenged this view. They found that substrate (DMADP)
was less available, but there was more activity of isoprene synthase at elevated
[CO2 ]. Possell and Hewitt (2011) found less DMADP and isoprene synthase
activity in plants grown in elevated [CO2 ] but their DMADP measurements were
made by acidifying whole leaves. This technique measures whole-leaf DMADP,
while only DMADP inside chloroplasts is readily available for isoprene production
and acidification was found to significantly overestimate DMADP levels when
compared to measurements made using mass spectrometry (Weise et al. 2013).

5.4.3 Isoprene Synthase Gene Expression

Much of the changes in long-term emission capacity are related to changes in the
expression of the isoprene synthase gene. Given that the typical substrate levels
in leaves are in the range of the Km , both IspS capacity and DMADP supply
rate will affect the overall rate, and DMADP supply is likely the most important
factor for short-term changes in rate of emission. Only one report has suggested
strong post-translational regulation of isoprene synthase (Lehning et al. 2001), but
in most studies this has not been invoked. Gene expression can be controlled by
many factors, but studies of the effect of the DNA immediately upstream of the
coding sequence (the promoter region) are just beginning. The promoter region of
142 Z. Li and T.D. Sharkey

grey poplar (Populus x canescens) IspS was sequenced and examined for motifs
known to influence gene expression (Loivamäki et al. 2007). A motif known to
confer circadian regulation was found. Similar circadian elements were reported
from Populus trichocarpa in IspS, DXS, CMS, MCS, and HDS by Wiberley et al.
(2009). Heat shock promoter elements were found in IspS, DXS, and HDR as well.
The grey poplar promoter was tested by fusing it to a reporter gene and expressing it
in Arabidopsis (Cinege et al. 2009). The promoter caused gene expression primarily
in leaves and had properties that would explain a number of properties of IspS
expression.
Many genes have a motif consisting of TATA that serves to start the process of
transcribing the DNA into RNA for subsequent protein production. In grey poplar
(Populus x canescens) this TATA box was proposed to reside about 100 base pairs
upstream of the protein coding start site, but a more likely TATA box is found 1270
base pairs upstream (Sharkey, T.D., unpublished data). On the other hand, only 250
base pairs of upstream sequence is available for kudzu (Pueraria lobata) IspS and
a possible TATA box is found near the beginning of this region. It was possible
to express this kudzu gene in Arabidopsis using this short promoter, providing
evidence that the TATA box is functional (or that some other transcriptional start
motif is used in the case of kudzu). Soybean (Glycine max) has two genes nearly
identical to the kudzu IspS, but soybean does not emit isoprene. The promoter
regions of the two soybean genes show significant alteration and lack the possible
TATA box found in kudzu (Sharkey, T.D., unpublished data). There are also no
reported ESTs for these genes suggesting that these are pseudogenes that are no
longer expressed. These provide insight into how plants have lost the trait of
isoprene emission. The analysis of the promoters is now much easier because of
large-scale sequencing projects. Important gene expression controls can be studied
and new insights are likely to come in the near future (Rajabi Memari et al. 2013;
Rosenkranz and Schnitzler 2013 for further discussion).

5.5 Conclusions and Future Perspectives

Significant advances are being made on the front of understanding biochemical

and molecular regulation of BVOC emissions. The elucidation of MEP pathway
enzymes in the early 2000s, the new techniques developed in measuring trace
BVOC levels (e.g., fast isoprene sensor, proton-transfer reaction time-of-flight mass
spectrometer) and the advent of the omics era all contributed to the increasing
repertoire of knowledge about biochemical and molecular regulation of BVOC
emissions from trees. In particular, the potential for using the MEP pathway in
bacteria as chemical factories for producing commercially profitable compounds,
as well as targeting the bacterial MEP pathway in drug development, has sparked
tremendous interest and studies to elucidate the control mechanisms of the MEP
pathway. However, caution should be taken as we extrapolate existing knowledge
about the MEP pathway in microorganisms to understand BVOC regulation in
5 Molecular and Pathway Controls on Biogenic Volatile Organic Compound. . . 143

trees, as the two systems may not be entirely identical. A notable example, as
mentioned above, is HDS. The plant HDS enzyme accepts electrons directly from
photosynthesis (ferredoxin rather than NADPH) and may be an important regulatory
step in nature. The bacterial homolog, IspG, on the other hand, uses NADPH as
the source of reducing power. The MEP pathway metabolic profile of E. coli also
appears to be distinct from that of plant extract (Weise, S.E., Li, Z., Sharkey, T.D.,
unpublished data). Nevertheless, significant additional progress in understanding
molecular and biochemical control of BVOC emission from trees is likely in the
near future.

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Chapter 6
Metabolic and Gene Expression Controls
on the Production of Biogenic Volatile
Organic Compounds

Russell K. Monson

Abstract The emission of biogenic volatile organic compounds (BVOCs), in-

cluding isoprenoid compounds, methanol and oxygenated organic compounds, is
controlled by both the existing metabolic potential of a leaf and gene expression
responses that modulate the existing metabolic potential to increase or decrease
compound biosynthesis and emission rate. This capability to respond both instan-
taneously and in the long term to environmental variation provides plants with
flexibility in their adaptions to biotic and abiotic stresses, which are also encountered
in short and long-term time frames. This chapter reviews the mechanistic basis
of the immediate controls of volatile BVOC emissions by light, temperature, and
ambient CO2 and O2 concentrations, as well as the genetic responses that involve
changes in gene expression patterns. Photosynthesis ultimately provides the carbon
for BVOC production, though under non-stressed conditions the photosynthetic rate
itself is rarely so low that it limits BVOC emissions. However, various metabolic
pathways compete for substrates that are produced from photosynthate, including
cytosolic pathways, such as the mevalonic acid (MVA) pathway and chloroplastic
pathways such as the 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-
phosphate pathway (MEP/DOXP). Controls over the use of substrate are regulated
among these pathways through feedback mechanisms, specificity in the transport of
metabolites across organelle membranes, and the channeling of NADPH reductant
and ATP to specific steps in the pathways. This chapter emphasizes that these
interactive controls provide the major explanation for longer-term physiological
controls of emissions. Emissions of several types of compounds are considered,
including isoprenoids, methanol, and green leaf volatiles such as various aldehydes
and ketones.

R.K. Monson ()

School of Natural Resources and the Environment and the Laboratory for Tree Ring Research,
University of Arizona, Tucson, AZ 85721, USA
e-mail: [email protected]

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 153
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 6,
© Springer ScienceCBusiness Media Dordrecht 2013
154 R.K. Monson

6.1 Introduction

The broad diversity of biogenic volatile organic compounds (BVOCs) emitted by

plants reflects the flow of carbon substrates through a diverse set of metabolic
pathways (Gershenzon and Kreis 1999; Dudareva et al. 2005). This chemical
diversity is required to provide plants with the ability to mitigate the influence of
biotic and abiotic stresses and capture the resources required to maximize growth,
competitive persistence, and ultimately, reproductive fitness (Fineschi et al. 2013).
However, with access to a diversity of chemical options comes the challenge to
regulate how that diversity is expressed in the face of a dynamic environment
that changes across multiple timescales, and how to efficiently leverage existing
processes and pathways to produce novel opportunities for plant adaptation as novel
environments are encountered.
In the case of BVOCs, the evolutionary process has often co-opted and modified
preexisting metabolic pathways as a means to produce compounds with novel
structures and functions (Fineschi et al. 2013). Thus, many of the pathways that
produce BVOCs are connected through mutual and parallel reliance on intermediate
metabolites (pathway ‘cross-talk’) and the serial channeling of metabolites from
one pathway to another (Lichtenthaler 1999; Bick and Lange 2003; Hampel et al.
2005; Hemmerlin et al. 2012). Control over the flux of metabolites through these
pathways requires mechanisms for irreversibility in key catalyzed steps, feedbacks
to regulate the activity of an entire pathway, and allosteric modification of specific
enzymes to adjust the catalytic potential to process metabolites. Furthermore, many
pathway products and intermediate metabolites are channeled among different
cellular organelles, providing an even greater number of possibilities for the
compartmentation and control over metabolic flux. In addition to these direct
interactions among metabolic components of the cell, control over the production
and emission of BVOCs is accomplished by constitutive and inducible genetic
processes that regulate the timing and amount of enzyme catalysts produced. Many
of these genetic controls are, in turn, regulated by cascades of metabolic effects that
respond to ontogenetic or environmental cues (Keeling and Bohlmann 2006; Li and
Sharkey 2013).
Taken together, these controls must be responsive to short-term (seconds-to-
hours) and long-term (hours-to-weeks) changes in the environment, and they must
reflect an interface between the perception of changing environmental cues and
adjustments in the activity and expression of metabolic potential. In this chapter,
I will focus on the mechanisms by which BVOC production and emission is
controlled in short- and long-term scenarios at the cellular, leaf and plant scales.
Most of the focus of my discussion will be on the production and emission of
isoprenoid BVOCs, because most past research has focused on these compounds
and the pathway controls are known to the greatest extent. I will also take up
the issue of controls over the production and emission of methanol, acetaldehyde
and other oxygenated BVOCs. I will focus on research that has concerned BVOC
emissions from trees and forests, as once again this has been the subject of most
past research.
6 Metabolic and Gene Expression Controls on the Production of Biogenic. . . 155

6.2 The Short- and Long-Term Controls over BVOC

Emissions with Emphasis on Isoprene

Conventionally, the emission of BVOCs has been defined according to the metabolic
potential available for compound biosynthesis that can be influenced by instan-
taneous changes in the environment (the instantaneous emission rate) and the
overall capacity for compound biosynthesis that is independent of instantaneous
changes in the environment (the basal emission rate, or emission factor). This
type of classification was historically developed for the case of isoprenoid BVOCs
(Guenther et al. 1991, 1993). Partitioning of the BVOC emission rate into these
two levels of control has not only served our conceptual understanding of cellular
processes (the instantaneous rate is principally controlled by the availability of
substrate or kinetic constraints of rate-limiting enzymes and the basal rate is
principally controlled by gene expression and limiting enzyme activity), but it
also provided a convenient structure by which to design BVOC emission models
(Monson et al. 2007, 2012 for reviews).
Early in the writings on isoprenoid emissions the concepts of ‘instantaneous and
basal control’ were equated to ‘short-term and long-term’ control, respectively (e.g.,
Monson et al. 1995). This conflation of ‘types of control’ with ‘timing of control’
was, in hindsight, unfortunate. We now know that changes in gene expression can
under exceptional circumstances have an influence on BVOC emission rates on the
timescale of hours (Wilkinson et al. 2006; Loivamäki et al. 2007; Wiberley et al.
2009) (traditionally considered ‘long-term control’), i.e., almost the same timescales
by which substrate limitations can develop (Magel et al. 2006; Li et al. 2011; Li and
Sharkey 2013) (traditionally considered ‘short-term control’). Thus, the traditional
metrics used to differentially classify these responses are not reliable. While some
responses, such as the light- and temperature dependence of isoprene emissions
can clearly be differentiated into instantaneous (short-term) and basal (long-term)
processes, classification along these lines has become more tenable than previously
thought. In recognition of these difficulties, in this chapter, I will refer to metabolic
responses as those that occur through dynamics to metabolite fluxes and pathway
interactions (what might previously have been referred to as ‘short-term responses’)
and I will refer to gene-expression responses as those reflected by a change in
gene product levels (what might previously have been referred to as ‘long-term
responses’) (Fig. 6.1).
Several examples serve to illustrate these distinctions further. The responses of
isoprene emission rate to temperature (Magel et al. 2006; Rasulov et al. 2010),
incident PPFD (Rasulov et al. 2009a) and changes in CO2 concentration (Rosenstiel
et al. 2003; Rasulov et al. 2009b; Trowbridge et al. 2012; Calfapietra et al.
2013) are due to dynamics in substrate concentration and perhaps the kinetic
properties of the isoprene synthase enzyme. Similarly, studies that have transferred
the isoprene synthase gene into otherwise non-emitting species have shown that
the instantaneous emission rate is limited by the existing capacity to produce the
immediate isoprene synthase substrate dimethylallyl diphosphate (DMADP) within
156 R.K. Monson

instantaneous emission rate

metabolic responses
prevailing leaf temperature
substrate availability
prevailing PPFD
enzyme kinetics
prevailing intercellular CO2 concentration

environmentally-altered basal emission rate

gene-expression responses
climate history (e.g., drought, temperature)
gene transcription and
mechanical injury (e.g., fungal infection)
translation
herbivory

basal emission rate

Fig. 6.1 A general scheme showing how gene-expression responses and metabolic responses
combine to produce an instantaneous BVOC emission rate that is dependent on both longer-term
and shorter-term cues and influences of the environment

the target plant (Sharkey et al. 2005; Vickers et al. 2011). In other words, transfer
of the enzyme gene is not accompanied by an upregulation of the pathway that
produces the substrate, and the resulting substrate limitation is a simple result of the
transferred enzyme activity being greater than the existing potential for substrate
production. All of these examples can be classified as control due to metabolic
response.
In contrast, control by gene expression response infers that past natural selection
has favored control over the activity of specific genes in a way that causes a change
in the metabolic potential to synthesize and emit compounds. For example, in the
case of terpenes that deter herbivory or infection, biosynthesis potential may need
to be upregulated in coordination with specific phenological phases of the plant
(e.g., Huang et al. 2012) or after being induced by episodes of injury (Schiebe
et al. 2012). These types of control often involve complex signalling pathways in
the plant, leading to specific patterns of gene expression. These are all examples of
what I classify as control by gene expression. Specific cases of this type of control
have been described in the initiation of isoprenoid emission capacity during leaf
maturation (Wiberley et al. 2005), shifts in emission capacity during acclimation to
past temperature regimes (Cinege et al. 2009; Wiberley et al. 2009), modifications
in emissions following exposure to ozone (Calfapietra et al. 2007; Fares et al. 2010;
Loreto et al. 2004), increases in emissions in response to transitions from lower
versus higher growth light regimes (Cinege et al. 2009).
6 Metabolic and Gene Expression Controls on the Production of Biogenic. . . 157

6.2.1 Pathways and Controls for Substrate Channeling

to Terpenoid BVOCs

Prior to 1997, a general consensus existed in the BVOC research community

that carbohydrates produced through recent photosynthesis were channeled to the
production of dimethylallyl diphosphate (DMADP) and its isomer, isopentenyl
diphosphate (IDP) through the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) re-
ductase pathway, or mevalonic acid (MVA) pathway (also called the Bloch-Lynen
pathway in the animal metabolism literature) in the cytosol of cells (Gray 1987 for
a review). The incorporation of 13 C-labeled glucose into IDP was clearly observed
in studies of cell-free extracts in mammals, fungi, bacteria and plants (Spurgeon
and Porter 1981 for a review). In the MVA pathway, three molecules of acetyl-CoA
are condensed to HMG-CoA, which is then chemically reduced to the six-carbon
compound, mevalonic acid. Mevalonic acid, in turn, is converted to IDP with an
energetic cost of 3 ATP molecules and loss of one CO2 molecule.
In the mid-1990s, however, tracer studies in bacteria and algae revealed that not
all taxa incorporated 14 C label into IDP in exactly the same way (Horbach et al.
1993; Rohmer et al. 1996; Schwender et al. 1996). Furthermore, it was difficult
to reconcile observations that 13 CO2 that was fed to leaves was observed in some
emitted BVOCs, like isoprene, over the timescale of seconds, yet metabolite pools
in the MVA pathway turned over relatively slowly, on the timescale of minutes
(Delwiche and Sharkey 1993). In early 1997, Lichtenthaler et al. proposed that two
distinct pathways exist in plants for the synthesis of DMADP and IDP, with one
being the traditional MVA pathway in the cytosol and one being a novel pathway,
which is now recognized as the 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-
D-xylulose 5-phosphate pathway (MEP/DOXP) pathway in the plastids (Li and
Sharkey 2013; Rosenkranz and Schnitzler 2013). Studies using deuterium-labeled
intermediates verified that isoprene is produced in the chloroplast with substrate
provided by this novel pathway (Zeidler et al. 1997). Thus, within 3–4 years, a
rapid series of discoveries allowed us to better understand how recent photosynthate
is tightly coupled in time with the production of at least some terpenoid BVOCs,
and to identify the types of controls that regulate carbon channeling during BVOC
production (Fig. 6.2).
Discovery of the new, pathway, however, did not explain the origin of all BVOCs,
as some, such as the C15 sesquiterpenes, could only be produced by terpene
synthase enzymes localized in the cytosol; at the time of discovery of the MEP
pathway there did not appear to be a mechanism for exporting IDP from the
chloroplast (Gershenzon and Kreis 1999). Thus, sesquiterpene biosynthesis appears
to be produced principally by substrate provided by the MVA pathway. We now
recognize that there is a significant amount of metabolite exchange between both
pathways, and that compensatory control mechanisms exist to balance the activities
of the two pathways (Dudareva et al. 2005; Nagegowda 2010; Hemmerlin et al.
2012). Furthermore, distinct cytosolic and plastidial sesquiterpene synthases have
been demonstrated (Nagegowda 2010; Rajabi Memari et al. 2013).
158 R.K. Monson

citrate MVA pathway

acetate
acetyl-CoA IDP
TCA cycle

sesquiterpenes DMADP IDP

monoterpenes

monoterpenes

pyr pyr
terpene synthase
other
biosynthetic PEP PEP
pathways
pyr MEP pathway DMADP
glycolysis IDP
GAP

isoprene synthase

photosynthesis
isoprene
cyt chl

Fig. 6.2 The relationships between the 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose

5-phosphate pathway (MEP/DOXP pathway) and mevalonate (MVA) pathway in synthesizing
isoprene and monoterpenes in the chloroplast and sesquiterpenes and monoterpenes in the cytosol.
The potential for ‘cross talk’ between the pathways occurs through exchange of the common
metabolite isopentenyl diphosphate (IDP) between chloroplasts (chl) and the cytosol (cyt)

It is worth noting at this point that there is strong evidence, beyond tracer
labelling studies, that isoprene is produced solely in the MEP pathway. A cytosolic
form of isoprene synthase (IspS), the enzyme that catalyzes isoprene formation from
DMADP, has not been discovered; leaving its possible production from MVA path-
way metabolites undefined. Using immunogold-labelling with polyclonal antibodies
generated against recombinant isoprene synthase protein, Schnitzler et al. (2005)
showed that in poplar leaves this enzyme is located in the chloroplast stroma, with
some fraction of the protein attached to the outside (stromal) surface of thylakoid
membranes; this is consistent with previous studies showing both soluble and
membrane-bound fractions of isoprene synthase in willow leaf extracts (Wildermuth
and Fall 1998). Furthermore, in transgenic tobacco (Nicotiana tabacum) which has
been engineered to express isoprene synthase in the cytosol, no significant amounts
of isoprene are emitted (Vickers et al. 2011).

6.2.2 Cross-Talk Between the Two Pathways for Isoprenoid

Biosynthesis in Plants

Plants are different than most organisms in that they utilise both the MEP and MVA
pathways, in parallel, for the biosynthesis of isoprenoid molecules. Most Eubacteria
6 Metabolic and Gene Expression Controls on the Production of Biogenic. . . 159

and Cyanobacteria utilise only the MEP pathway, whereas Archaea, and some
other Eubacteria such as staphylococci, streptococci and enterococci belonging to
Firmicutes utilise only the MVA pathway (Proteau 1998; Smit and Mushegian 2000;
Trutko et al. 2004). Streptomyces spp. (Actinobacteria) appear to have operon genes
for both pathways, and species in this group express both sets of genes, but in serial
manner at different stages of growth (Seto et al. 1996); not simultaneously as in
plants. Thus, one of the principal challenges faced by plant biologists has been to
discover the means of proper coordination of activities in the two pathways.
Until the past decade or so, it was assumed that compartmentalization of
isoprenoid biosynthesis was determined by the differential localization of ter-
pene synthase enzymes with hemiterpene (isoprene), monoterpene and diterpene
synthases occurring in the plastid, and sesquiterpene synthases occurring in the
cytosol. There was little interaction suspected for the exchange of substrate between
plastids and the cytosol. However, experiments in which deuterium-labeled 1-deoxy
D-xylulose (an intermediate metabolite of the MEP pathway) or fosmidomycin
(an inhibitor of the MEP pathway) were fed to cut snapdragon flowers, showed
that the MEP pathway provides substrate in the form of IDP to both the plastidic
and cytosolic synthesis of some BVOCs (Dudareva et al. 2005). The use of labeled
metabolites allowed Dudareva et al. (2005) to distinguish a one-way transport of
IDP from the plastid to the cytosol. The production of IDP to support BVOC
emissions in both pathways may be under circadian control. A circadian dependency
of MEP pathway activity has been demonstrated for the incorporation of labeled
intermediates into monoterpenes and sesquiterpenes in flowers (Dudareva et al.
2005). Also some circadian effects were observed in the emission of isoprene from
oil palm (Elaeis guineensis) leaves (Wilkinson et al. 2006) and in expression of
isoprene synthase gene in hybrid poplar leaves (Loivamäki et al. 2007). Thus,
a picture is emerging in which terpenoid BVOC emissions are synchronized by
circadian rhythms to photosynthetic activity by upregulating both the pathway to
produce substrate and the enzymes to produce a volatilized product. In a separate
set of studies, a combination of pathway inhibitors was used to identify Arabidopsis
mutants that could upregulate MVA pathway activity and survive in the presence
of fosmidomycin (i.e., in the absence of MEP pathway activity) (Rodrı́guez-
Concepción et al. 2004). One mutant was identified that appeared capable of
importing substrate (presumably in the form of IDP) from the cytosol into the
chloroplast for the production of carotenoids, chlorophylls and other critical primary
components of the photosynthetic system. Thus, there is now good evidence that,
while plants rely on two separate pathways for the production of BVOCs, they also
synchronize activities of the pathways, depending on their differential capacities to
produce IDP (Bick and Lange 2003). It has been argued that this type of interactive
control provides advantages to plants in being able to maintain homeostasis in the
face of different cellular demands on photosynthate and the isoprenoid precursors
derived from photosynthate (Hemmerlin et al. 2012).
Cross-talk between the MEP and MVA pathways may be part of a general
metabolic control that ensures the flow of substrate to pathways in the face of
variable photosynthate production. For example, Rontein et al. (2002) observed
160 R.K. Monson

that in cultured tomato (Solanum lycopersicum) cells grown with a finite supply of
glucose, glycolytic flux remained approximately constant as the available glucose
was depleted from 70 to 40 % of the supply. This stability in glycolytic flux was
not achieved by metabolic control within glycolysis, but rather by adjustments
to intersecting pathways that utilise glycolytic intermediates. Thus, under condi-
tions of replete carbohydrate, when glycolytic substrates were readily available,
intersecting pathways were up-regulated to pull intermediate metabolites out of
the central trunk of glycolysis, and increase the potential to synthesize secondary
products. Conversely, as glucose supply decreased, intersecting pathways were
down-regulated to preserve higher fluxes of intermediate metabolites through the
main trunk of the pathway. This concept of ‘network rigidity’ has been proposed
as a central tenet of metabolic adaptation (Stephanopoulos and Vallino 1991). In
the case of glycolysis, a principal adjustment appears to occur in regulation of the
availability of phosphoenol pyruvate (PEP), a glycolytic metabolite that also serves
as the substrate for amino acid biosynthesis and for anapleurotic compensation of
substrates in the tricarboxylic acid cycle. PEP is also a possible way through which
cytosolic metabolites can enter chloroplastic isoprenoid synthesis pathway. Once
transported to chloroplast, PEP can be converted to pyruvate, and thus enter the
production of BVOCs from the MEP pathway. Within the context of these relations,
it is reasonable to propose that photosynthetic activity and the availability of sugars
can be a control point around which the flow of pyruvate to the MEP pathway is
regulated.

6.2.3 Feedback Homeostasis in MEP Pathway Activity

6.2.3.1 Isoprene Synthase or Substrate Limitation?

One of the fundamental issues in need of clear understanding is the degree to which
the rate of biosynthesis of isoprenoid BVOCs is controlled by the activity of terpene
synthase enzymes or the availability of the two principal MEP pathway substrates –
glyceraldehyde 3-phosphate (GAP) and pyruvate (Pyr). In a study of isoprene
emission from kudzu (Pueraria lobata) leaves, Wolfertz et al. (2003) observed an
inverse correlation between the isoprene emission rate and chloroplast DMADP
concentration among 15 different leaves. This evidence was used to conclude that
substrate concentration does not control the instantaneous isoprene emission rate in
kudzu, but rather it is controlled by isoprene synthase activity. This conclusion is
based on the reasoning that if DMADP limited isoprene emission rate, the leaves
with the lowest emission rates should have also exhibited the lowest DMADP
concentrations. The analysis provides a strong line of evidence for primacy in the
role of isoprene synthase activity in controlling the isoprene emission rate. However,
there is potential for weakness in certain conclusions derived from the analysis.
The chloroplast DMADP concentrations used in the analysis were determined on
a sample of all five leaves with the lowest versus highest emission rates. Thus,
6 Metabolic and Gene Expression Controls on the Production of Biogenic. . . 161

Relative isoprene emission rate

1.00 40

Leaf temperature (⬚C)

leaf T

0.75 35

labeled DOX
30
0.50
25
0.25

0
1 2 3 4 5 6
Time of experiment (h)

Fig. 6.3 Time-dependent labelling of emitted isoprene by di-deuterated deoxyxyulose (DOX), the
first product formed in the MEP pathway. The thin dashed line shows an experimental progression
of leaf temperature through a stepwise treatment. The solid black line is the total isoprene emission
rate. The solid grey line is unlabeled emitted isoprene, and the dashed grey line is deuterium
labeled emitted isoprene. The results show a compensatory tradeoff between labeled and unlabeled
isoprene as a function of time since the application of the DOX label, suggesting feedback
regulation of the channeling of DOX into isoprene in the MEP pathway. The grey-shaded boxes
show periods of darkness before and after the experiment (Modified from Wolfertz et al. 2003)

it is not possible to assess whether the observed correlation was well distributed
across the 15 leaf samples – outlier values for a more limited number of leaves may
have over-influenced the correlation. There are also concerns about the accuracy of
separating the cytosolic and chloroplastic DMADP pools in these types of analyses;
the cytosolic pool of DMADP vastly exceeds the chloroplastic pool (Rasulov et al.
2009a), making accurate estimation of the latter difficult.
In an effort to better distinguish the relative levels of control by substrate avail-
ability versus enzyme activity, Wolfertz et al. (2003, 2004) fed leaves exogenous di-
deuterated deoxyxylulose (DOX), the first metabolite produced in the MEP pathway
(Fig. 6.3). Through this approach, these researchers tested the hypothesis that by
artificially increasing the flow of carbon to DMADP, isoprene emission rate would
be increased; this would support a role for substrate limitation. While it was shown
that the labeled exogenous DOX replaced unlabeled endogenous DOX in the emitted
isoprene, the overall rate of isoprene emission did not increase. Furthermore, at
high levels of exogenous DOX supply, the incorporation of endogenous DOX into
isoprene decreased to zero. The authors interpreted these results as indicating that
DMADP supply is controlled by end-product (DMADP) feedback. Once again,
these studies supported the lack of a role for substrate limitation in determining the
isoprene emission rate. A similar result that is consistent with end-product inhibition
was obtained by Ghirardo et al. (2010) using RNA interference (RNAi) techniques
to knock out production of the isoprene synthase enzyme in selected poplar lines.
These researchers observed reduced activities of deoxyxylulose phosphate synthase
(DXS), the first enzyme in the MEP pathway, in RNAi trees, compared to wild type
162 R.K. Monson

trees. However, they did not measure leaf DMADP levels to validate a correlation
between increases in end-product concentration and inhibition of DXS. In a separate
study, Behnke et al. (2010) used the same RNAi lines of poplar and observed
increases in leaf DMADP through the growing season in RNAi trees, compared to
wild type trees. This study provides indirect evidence that the end-product inhibition
hypothesized by Ghirardo et al. (2010) does indeed exist in the RNAi lines. In the
Behnke et al. (2010) study there was no observed downregulation of genes in the
initial steps of the MEP pathway. Thus, any end-product inhibition that might exist
is likely due to post-translational allosteric modification of DXS.

6.2.3.2 Limitations at Different Temperatures

Returning to the experiments of Wolfertz et al. (2003), the addition of exogenous,

labeled DOX did not result in significantly higher rates of labeled isoprene emission
at moderate leaf temperatures (30 ı C), but it significantly increased both the rate
of isoprene emission and fraction of isoprene labelling at higher leaf temperatures
(45 ı C). These results led the authors to conclude that while substrate limitations
may not be significant at moderate leaf temperatures, they are significant at higher
leaf temperatures. Temperature dependence in the control over isoprene emission
by substrate availability versus the kinetic constraints on isoprene synthase, as
revealed by Wolfertz et al. (2003), may explain the results of Fortunati et al. (2008).
In the latter study, poplar (Populus alba) trees which were exposed to extreme
drought stress and then allowed to recover, lacked a significant response in isoprene
emission rate when measured at 35 ı C, compared to 25 ı C. The kinetic response of
isoprene synthase in recovered leaves should have produced significantly higher
post-recovery emission rates; this result was not observed. Consistent with the
results of Wolfertz et al. (2003), it is possible that limited substrate availability
prevented isoprene synthase from reaching its potential catalytic activity at the
higher leaf temperature (35 ı C). Feedback control over the MEP pathway is shown
within the context of the metabolic and gene expression controls over isoprene
emission rate in Fig. 6.4.
A similar conclusion as to the role of differential temperature-dependent con-
straints on isoprene synthase activity has been reached in studies by Rasulov
et al. (2010). Using a post-illumination burst of isoprene emission to model and
quantify the chloroplast DMADP concentration, these researchers demonstrated that
at temperatures up to 30 ı C, the temperature response of isoprene emission rate was
not limited by DMADP substrate availability, but rather by kinetic constraints on
isoprene synthase activity. At temperatures greater than 30 ı C, however, isoprene
emission rate was influenced by both substrate availability and isoprene synthase
activity. In other studies by Magel et al. (2006) and Li et al. (2011), DMADP was
also observed to limit the temperature dependence of isoprene emission rate, but in
these cases, only at temperatures above 35 ı C and only after one hour.
6 Metabolic and Gene Expression Controls on the Production of Biogenic. . . 163

isoprenoid BVOCs

metabolic
isoprenoid synthase activity
control
other isoprenoid synthase content
biosynthetic PEP PEP (−)
pathways
MEP pathway activity
pyr DMADP
glycolysis IDP
GAP MEP pathway content

photosynthesis gene expression

control
cyt chl

Fig. 6.4 Scheme showing end-product (DMADP) control over the flux of substrate through the
MEP pathway within the context of gene-expression and metabolic controls over the isoprene
emission rate from chloroplasts (chl) and interactions with processes in the cytosol (cyt)

Consistent with all of these past observations, studies using 13 CO2 as a tracer
of recently-assimilated carbon in isoprene-emitting leaves have shown that while
leaves at all temperatures rely to some extent on older, stored carbon for substrate,
this reliance increases at higher leaf temperatures (Funk et al. 2004). In these
conditions, the rate of DMADP depletion by isoprene synthase activity may exceed
the rate by which photosynthesis can provide substrate for DMADP repletion.
Thus, substrate limitations to isoprene emission rate appear to increase when CO2
assimilation rate is also limited. Under these conditions, internal mobilization of
stored carbon (or injection of exogenously added carbon) compensates for the
substrate limitations.
The conclusion that the kinetic constraints on isoprene synthase limit the overall
emission rate, except at higher temperatures is generally dependent on the balance
between isoprene synthase activity and photosynthesis rate. When the catalytic
capacity of isoprene synthase is high and the capacity for GAP production through
photosynthesis is low (e.g., at high leaf temperatures or during drought), and in
particular under low light (Rasulov et al. 2009b), then the availability of DMADP
will have a greater role in limiting the emission rate. Similarly, when the capacity
for GAP production is high and the catalytic capacity of isoprene synthase is low
(either because of kinetic constraints or because of low isoprene synthase protein
concentrations), then isoprene synthase activity will have a greater role in limiting
the overall emission rate. This latter condition explains the inverse correlation
between isoprene emission rate and DMADP concentration observed in the study
of Wolfertz et al. (2003) that were discussed above.
164 R.K. Monson

6.2.3.3 Limitations at Different CO2 Concentrations

These same relations cannot explain the potential role of DMADP substrate
limitations when leaves are exposed to varying intercellular CO2 concentrations
(Sanadze 1964; Monson and Fall 1989; Loreto and Sharkey 1990; Rosenstiel et al.
2003; Centritto et al. 2004; Rapparini et al. 2004; Scholefield et al. 2004; Pegoraro
et al. 2005; Wilkinson et al. 2009; Possell et al. 2005; Possell and Hewitt 2011).
In that case, increases in intercellular CO2 concentration, even at temperatures
below 35 ı C, cause in increase in the capacity to produce GAP through greater
photosynthesis rates, but a decrease in the isoprene emission rate. There is no
evidence to date of a CO2 -dependent, direct effect on isoprene synthase kinetics.
However, leaf DMADP concentrations have been shown to decrease as intercellular
CO2 concentration increases (Rosenstiel et al. 2003; Rasulov et al. 2009b; Sun et al.
2012). Thus, DMADP substrate availability appears to be the primary cause of the
CO2 -dependency of isoprene emission rate, despite a high ratio in the capacity to
produce GAP substrate relative to the activity of isoprene synthase. In this case, the
substrate limitation appears to be due to either the rate at which pyruvate is supplied
to the chloroplast (Rosenstiel et al. 2003) or the rate at which GAP is converted to
DMADP in the chloroplast (Rasulov et al. 2009b). In the past (Monson et al. 2012),
I have favored the hypothesis offered by Rosenstiel et al. (2003) because of results
obtained by Trowbridge et al. (2012). In the latter study, proton-transfer reaction
mass spectrometry was used to detect the differential kinetics of 13 C incorporation
into fragments of isoprene presumed to come from cytosolic versus chloroplastic
sources (Fig. 6.5). The results during periods of low versus high intercellular CO2
concentration suggested slower labelling in the fragment purported to come from
cytosolic sources (which provides pyruvate to the chloroplast), and this fragment
was more highly labeled in the presence of low CO2 , compared to that derived from
GAP directly (which would not be expected if chloroplastic ATP limited the overall
emission rate). These latter results can be interpreted as supporting the Rosenstiel
et al. (2003) perspective more than the Rasulov et al. (2009b) perspective.

6.3 Short- and Long-Term Controls Over Emissions

of Other BVOCs

6.3.1 Monoterpenes

Monoterpene emissions from leaves and needles are subject to both metabolic
and gene expression controls. In coniferous species, monoterpenes tend to be
stored as a component of oleoresin, a viscous solution of terpenes, acids and
phenolic compounds that can be stored in individual cells, ‘blister-like’ structures,
or continuous duct systems (Lewinsohn et al. 1991). In this chapter, I will focus
on terpene emissions from resin ducts, which are most iconically represented
in the genus Pinus. The terpene composition of oleoresin is largely determined
6 Metabolic and Gene Expression Controls on the Production of Biogenic. . . 165

7
a
6
5

4
Emission rate (nmol m−2 s−1) 3
2
1

b
6

4
3

0
0 750 1500 2250 3000
Time (seconds)

Fig. 6.5 Labelling of carbon atoms using assimilated 13 CO2 in mass 69C (M69C ) and mass 41C
(M41C ) and their isotopomers through time. (a) 13 CO2 -labelling of carbon atoms in trees grown
and measured in ambient CO2 conditions (400 mol mol1 CO2 ) in the parent isoprene molecule,
as characterized by a decrease in the M69C signal (orange circles) and simultaneous increase
in its isotopomers (denoted as sums) as labeled carbons were successively incorporated through
time. Total emission (blue circles), M70C (red downward triangles), M71C (green triangles),
M72C (yellow squares), M73C (sea green squares), M74C (purple diamonds) are represented. (b)
13
CO2 -labelling of carbon atoms in trees grown and measured at 30 ı C in ambient CO2 conditions
(400 mol mol1 CO2 ) in the 3-C-methyl-vinyl isoprene fragment, characterized by a decrease
in the M41C signal (light orange dotted downward triangles) with a simultaneous increase in its
labeled isotopomers (denoted as sums). Total emission (blue dotted squares), M42C (pink crossed
circles), M43C (green hexagons), M44C (yellow diamonds) are represented. Before leaves were
exposed to 13 CO2 -labelling at 1,000 s, plants were exposed to the same 12 CO2 concentrations at
which they were grown. The simultaneous labelling of the first carbon in the parent molecule
(M70C ) and the fragment (M42C ) suggest that the first carbon contributing to the synthesis of
isoprene comes from the M41C fragment. However, while all of the isoprene molecules show the
next two carbons labeled shortly after (M71C and M72C ), the next two carbons on the M41C
fragment (M43C and M44C ) are never fully labeled and may result from the incomplete labelling
of pyruvate

by genetic control; however, environmental variation can also induce chemical

variation (Nerg et al. 1994; Keeling and Bohlmann 2006; Nikolic et al. 2008). The
actual biosynthesis of the terpene components of oleoresin occurs in epithelial cells
that line the walls of the resin ducts. Monoterpenes can leave the liquid phase of
166 R.K. Monson

the oleoresin and diffuse in the vapour phase through the internal tissues of the
needles to stomatal pores, where they can be emitted to the atmosphere. Metabolic
controls are not significant in determining the rate of volatile terpene emission from
conifer needles, although physiological controls over diffusive resistance, including
stomatal resistance, and environmental controls such as temperature, can impose
significant control (Lerdau 1991; Lerdau and Gray 2003). Interactions among
needle temperature, volatility (the Henry’s law constants and octanol/water partition
coefficients) of the terpenes composing the oleoresin and diffusive resistance
represent what have traditionally been referred to as ‘short-term’ controls in pine
needle emission rates (Lerdau et al. 1994).
Needle herbivory imposes both physiological (diffusive) and genetic controls on
the monoterpene emission rate. In terms of the shorter-term, physiological control,
herbivory has the potential to make new breaks in the diffusive barriers to terpene
evaporation to the atmosphere (Litvak and Monson 1998; Loreto et al. 2000), thus
causing an immediate increase in the needle emission rate. In terms of genetic
control, herbivory causes an increase in the expression of genes that encode terpene
synthase enzymes in both needles (Litvak and Monson 1998; Martin et al. 2003) and
stems (Schmidt et al. 2011). These genes are often induced by complex signalling
webs involving plant hormones, such as jasmonic acid (van Poecke and Dicke 2004).
In many species, mechanical injury by herbivory or fungal infection can induce the
formation of additional ‘traumatic’ resin ducts in wood, a process that potentially
contributes to increased terpene emission (Christiansen et al. 1999; Martin et al.
2002), though the emissions from stems and branches to the atmosphere have been
poorly characterized. Many pine species also exude droplets of oleoresin at the
juncture of needle follicles and cone bracts during the spring, when resin pressures
are high. The exposure of oleoresin directly to atmosphere through exuded droplets
has the potential to significantly affect canopy emission rates (Eller et al. 2013).
In the past, it was assumed that most monoterpene emissions from pine needles
were from stored oleoresin pools. However, it has been shown using 13 CO2 -labelling
that in some species, such as Pinus sylvestris (Ghirardo et al. 2010; Shao et al.
2001) and Pinus pinea (Noe et al. 2006), 30–90 % of the observed monoterpene
emissions were derived from recently assimilated CO2 . These emissions are light-
dependent (Staudt et al. 1997; Niinemets et al. 2002), similar to light-dependent
monoterpene emissions observed in some broad-leaved species (Lerdau and Gray
2003 for a review), and result from the channeling of carbon substrate through
the MEP pathway. For this type of monoterpene emissions, the same metabolic
and genetic controls that were discussed above for isoprene emissions are relevant
(Owen et al. 2002).

6.3.2 Methanol

Leaf methanol emission is catalyzed by the enzyme pectin methylesterase (PME)

which is most active in the cell wall domain of plant cells (Micheli 2001; Pelloux
6 Metabolic and Gene Expression Controls on the Production of Biogenic. . . 167

et al. 2007). PMEs catalyze the demethylation and associated esterification of

homogalacturonic acids (HGA), which are used to compose the polysaccharide
matrix of the primary plant cell wall. The products of de-methylation include
methanol and protons. The HGA substrate for PMEs is secreted into the cell wall
domain and constitutes the pectin fraction of the cell walls. Short-term control over
the rate of methanol emission is determined by the availability of HGA substrate
(Oikawa et al. 2007) and interactions between the solubility of methanol in the
aqueous phase of the leaf and stomatal diffusion resistances to leaf-atmosphere
exchange (Niinemets and Reichstein 2003; Harley et al. 2007; Harley 2013). There
is evidence for light-dependency of methanol emissions, but this appears to be due
to the response of stomatal resistance to the incident photosynthetic photon flux
density (PPFD), rather than a direct effect of PPFD on substrate availability (Oikawa
et al. 2007; Harley et al. 2007). Several studies have shown light-dependency
in the acidification of the primary cell walls of leaves, which in turn enhances
leaf expansion rate through a variety of mechanisms (van Volkenburgh 1999).
Acidification can occur in as quickly as in a few minutes following an increase
in PPFD (Elzenga et al. 2000). The activity of PMEs in producing methanol is
highly sensitive to cell wall pH (Catoire et al. 1998). Thus, it is possible that
changes in PPFD could elicit changes in the rate of export of HGA from cells and/or
changes in the activity of PMEs, and thus elicit changes in methanol emission;
however, this response has not been detectable in recent experiments (Oikawa et al.
2007). Furthermore, nighttime acidification can occur in some species, and thus, a
significant fraction of diurnal methanol emissions can be stored internally at night
and released the following morning when stomata open (Hüve et al. 2007). Thus,
for the present time, it appears that changes in stomatal resistance are the principal
causes for light-dependency in the methanol emission rate, and even that level
of control is due to slow equilibration between aqueous- and gas-phase methanol
following a change in stomatal resistance (Harley 2013 in this volume).
Long-term controls over methanol emissions from leaves have not been explicitly
studied. However, given the evidence to date that methanol emission is the result
of PME activity and HGA availability, it is reasonable to propose that methanol
emission capacity is controlled by levels of expression in the genes encoding the
production of PMEs and the enzymes controlling the biosynthesis and/or export of
HGA. Secretion of HGA is most active during leaf expansion (Pelloux et al. 2007).
Thus, methanol emissions are the highest in maturing leaves (Nemecek-Marshall
et al. 1995). Damage to leaves by feeding caterpillars increases the rate of methanol
emission, and this response can be replicated by the treatment of cut leaves with oral
secretions from the caterpillars (von Dahl et al. 2006). It was discovered that the oral
secretions elicit a change in pH in the cell wall domain of damaged tissues, which
in turn caused an increase in the transcript levels for specific PMEs. The possibility
has been raised that methanol acts as an ecological signalling molecule that affects
plant-to-plant communication (Dorokhov et al. 2012).
168 R.K. Monson

6.3.3 Acetaldehyde and the ‘Green Leaf Volatiles’

The biosynthesis of a variety of oxygenated BVOCs in leaves has been linked

to both metabolic imbalances in the flow of carbon between glycolysis and
mitochondrial respiration, and wounding, which in turn induces the oxidation of
fatty acids (Loreto and Schnitzler 2010). One of the most frequently identified
oxygenated compounds emitted from leaves is acetaldehyde. Historically, studies
on acetaldehyde emission have focused on anoxia in the root zone (Kimmerer
and Macdonald 1987; Kreuzwieser and Rennenberg 2013). Under anoxia, aerobic
respiration is inhibited, and the pyruvate formed through glycolysis is oxidized to
ethanol plus CO2 in a two-step type of ‘fermentation’ catalyzed in the first reaction
by pyruvate decarboxylase (PDC) to form acetaldehyde plus CO2 , and catalyzed in
the second reaction by alcohol dehydrogenase (ADH) to form ethanol. The ethanol
is then transported through the plant xylem to the leaves, where it is oxidized
in a two-step process, first to acetaldehyde in a reaction catalyzed by ADH (the
reverse of the reaction in the root that produced the ethanol), then to acetate in a
reaction catalyzed by aldehyde dehydrogenase (ALDH) (Kreuzwieser et al. 1999).
It is presumed that relatively volatile acetaldehyde leaks from the leaf pool, between
the steps of ADH and ALDH. In support of this hypothesis, Graus et al. (2004)
fed disulfiram, an inhibitor of ALDH to leaves, and observed a marked increase in
acetaldehyde emissions.
In a second process, it has been shown that acetaldehyde is emitted as emission
bursts following rapid light–dark transitions in leaves, and this process is inde-
pendent of the oxygen status of roots (Karl et al. 2002; Graus et al. 2004; Brilli
et al. 2011; Jardine et al. 2012). Leaf mesophyll and vein tissues have constitutive
activities of pyruvate decarboxylase (Kimmerer and MacDonald 1987; Nguyen et al.
2009), so it is feasible that acetaldehyde is formed directly from pyruvate. Karl et al.
(2002) demonstrated that the observed bursts of acetaldehyde were not accompanied
by ethanol emissions, suggesting it as a product of PDC acting alone or in
sequence with ALDH (producing acetate as a final product). Tracer studies showed
that recently assimilated 13 CO2 in leaves was transferred into emitted aldehyde,
suggesting a process confined to the leaves (also see Jardine et al. 2012). Karl et al.
(2002) proposed that the oxidation of pyruvate to acetaldehyde might function as an
‘overflow’ mechanism, allowing the cell to balance the production of pyruvate by
glycolysis against the demands imposed by mitochondrial respiration. This balance
may be necessary because of the highly regulated nature of pyruvate dehydrogenase
(PDH), the ‘bridge’ between glycolytic production of pyruvate and the production of
acetyl-CoA, which enters mitochondrial respiration. According to this hypothesis,
during a rapid light-to-dark transition, leaf pyruvate concentrations increase due to
adjustments in the partitioning of glycolytic products and intermediates to several
pathways. The buildup of pyruvate presumably triggers an increase in the activity
of PDC, producing a burst of acetaldehyde synthesis (Fig. 6.6).
This hypothesis was tested by Graus et al. (2004) in an experiment in which a
combination of acetylphosphinate, an inhibitor of PDH, and disulfiram, an inhibitor
6 Metabolic and Gene Expression Controls on the Production of Biogenic. . . 169

Acetaldehyde

Photosyn

(leaked emission) GAP GAP

Chl
PEP PEP
CO2

Pyr
Acetaldehyde Pyruvate
Pyruvate
decarboxylase
Pyr
Pyr dehydrogenase
Acetate Acetyl-CoA
Aldehyde
dehydrogenase Mit
Respiration

Fig. 6.6 A scheme showing the possible origin of emitted acetaldehyde as an ‘overflow’ from
excess pyruvate that accumulates due to imbalances between its production in glycolysis and
consumption in the mitochondrial tricarboxylic acid (TCA) cycle. Chl stands for chloroplast and
Mit for mitochondrion

of ALDH, was fed to leaves of poplar (P. x canescens). Together, these inhibitors
should lead to reduced consumption of pyruvate by PDH (i.e., reduced production of
acetyl-CoA) and reduced consumption of acetaldehyde by ALDH, resulting in the
channeling of even more pyruvate than normal to an enlarged acetaldehyde pool. In
fact, this combination did indeed lead to an increase in acetaldehyde emissions from
leaves. However, when they put leaves treated with disulfiram through a light–dark
transition, they did not observe an increase in emissions relative to control leaves;
in fact, they observed a large decrease (Graus et al. 2004). From these results, it
was concluded that indeed elevated acetaldehyde pools in the leaf lead to increased
acetaldehyde emissions from the leaf, but that this is not the cause of the previously
observed light-to-dark bursts of emissions.
Instead, it was suggested that the acetaldehyde bursts observed during light-to-
dark transitions could be linked to the wound-related oxidation of fatty acids, a
process that releases so-called ‘green leaf volatiles’ (Graus et al. 2004). In fact, there
is evidence that some of the wound-induced ‘green-leaf’ volatiles (GLVs), such as
hexenyl acetate, are derived from pyruvate (Jardine et al. 2009; Jardine et al. 2012)
and acetate units released from the breakdown of membrane fatty acids following
wounding (Cojocariu et al. 2005; Loreto et al. 2006). The production of green leaf
volatiles requires oxygen in a process catalyzed by lipoxygenase enzymes. In past
170 R.K. Monson

studies, the emissions of GLVs from leaves have been attributed to various biotic
and abiotic stresses including ozone exposure (Heiden et al. 2003; Beauchamp
et al. 2005), freeze-thaw episodes (Fall et al. 2001; Copolovici et al. 2012), high
light and temperature extremes, as well as mechanical wounding (Loreto et al.
2006), and programmed cell death during senescence (Holopainen and Gershenzon
2010). Returning to the case of Graus et al. (2004) in explaining an alternative
source of emitted acetaldehyde during light-to-dark transitions, it was proposed that
acetyl-CoA reacts with GLVs, including C6 aldehydes, to form C6 acetates; the
acetaldehyde was hypothesized to be leaking from the acetyl-CoA pool during this
reaction. Thus, in the Graus et al. (2004) hypothesis, unlike the pyruvate overflow
hypothesis, acetaldehyde emissions are linked to the wound-response production of
GLVs during light-to-dark transitions.
There are other possible explanations of the results from Graus et al. (2004),
however. As stated above, a novel high-affinity pyruvate decarboxylase has been
identified in the vein tissues of leaves (Nguyen et al. 2009). It is possible that this
enzyme is involved in the direct conversion of pyruvate to acetaldehyde plus CO2 .
Increases in cytosolic pyruvate during inhibition of ALDH may force more of the
pyruvate to the veins, where it is decarboxylated. Alternatively, the pyruvate may be
emitted directly to the atmosphere, as has been observed by Jardine et al. (2010). In
one study, Jardine et al. (2012) observed that leaves of mesquite (Prosopis velutina)
emitted no C6 green leaf volatiles when exposed to light-to-dark transitions in
anoxic atmospheric conditions, but did emit large amounts of acetaldehyde. In
the study by Brilli et al. (2011), a burst of GLV emissions was observed after a
light-to-dark transition in the grass Dactylis glomerata, but with no accompanying
acetaldehyde emissions. Together, the results of these studies do not support the
conclusion of Graus et al. (2004) that the acetaldehyde and green leaf volatile
bursts following light-to-dark transitions are necessarily linked. There is clearly a
lot of uncertainty that continues to surround the controls over acetaldehyde and
GLV emissions from leaves, especially following light-to-dark transitions. There is
a general consensus that the source of these emissions involves perturbations in the
balance of carbon flows among different primary and secondary pathways, but the
exact nature of those imbalances will likely need to be resolved through studies of
processes in isolated organelles, metabolic-control modelling and re-construction of
pathways through transgenic manipulation.

6.4 The Anthropocene and New Variables in the Control

Over BVOC Emissions

The primary changes expected to the global environment over the next century
due to continued societal and economic development include increases in the
atmospheric CO2 concentration, drier tropical forests, warmer temperatures at the
mid-latitudes and a transition from native to managed forest ecosystems (IPCC
2007). Environmental changes such as these are likely to affect landscape BVOC
6 Metabolic and Gene Expression Controls on the Production of Biogenic. . . 171

emission rates through existing potential for genetic control, as well as slower
ecological changes in community composition. The latter type of change is beyond
the scope of this chapter. With regard to the former type of change, we might expect
BVOC emissions to generally increase in proportion to the frequency of extreme
weather events. Episodes of extremely high temperature and drought tend to trigger
mechanisms that improve tolerance of abiotic stress through BVOC production
(Loreto and Schnitzler 2010).
Land-use change due to human activities has created a potential to change land-
scape surface albedo and influence the Earth’s radiation budget. Forest ecosystems
tend to have a lower surface albedo than pasturelands and grasslands. More recently,
political and economic initiatives have included strategies to expand reliance on
short-rotation agroforests for the purpose of producing cellulosic biofuels and
enhancing atmospheric CO2 sequestration (Pacala and Socolow 2004; Canadell and
Raupach 2008). Such land-use changes have the potential to not only influence sur-
face albedo and canopy temperatures, but also the emission of BVOCs (Purves et al.
2004). Bioenergy species such as poplars (Populus spp.), eucalypts (Eucalyptus
spp.), reed grass (Phalaris arundinacea), pines (Pinus spp.) and oil palm (Elaeis
guineensis) emit large quantities of reactive BVOCs (e.g., Ashworth et al. 2012).
Changes in the atmospheric CO2 concentration will further alter the potential for
landscape emissions (e.g., Heald et al. 2009). Many of the changes we might expect
on a future Earth trade off against each other in complex ways, making it difficult to
predict the ultimate effects of global change on BVOC emissions (Owen et al. 2013).
For example, in tropical latitudes, the planting of forests on reclaimed pasturelands,
has the potential to: (1) increase CO2 extraction from the atmosphere (a ‘cooling
effect’), (2) increase rates of latent heat loss (a ‘cooling effect’), (3) increase the
formation of secondary organic aerosols and density of clouds (a ‘cooling effect’),
and (4) increase the production of tropospheric ozone (a ‘warming effect’). In the
opposite direction, however, forests will cause a lower surface albedo and associated
higher flux of sensible heat and long-wave radiation to the atmosphere (a ‘warming’
effect). The net result of these interactions will require analysis with comprehensive,
coupled land-atmosphere climate and chemistry models (Ashworth et al. 2013;
Kulmala et al. 2013).

6.5 General Conclusions

The emission of BVOCs from leaves can be understood within the context of a
combination of metabolic responses, in which existing metabolic potential responds
to changes in substrate availability and enzyme kinetics, and gene-expression
responses, in which the regulation of gene transcript number and subsequent
translation control the amount of metabolic machinery capable of producing and
consuming substrate. In general terms, the metabolic responses tend to reflect
the shorter-term (seconds-to-minutes) responses, whereas the gene-expression re-
sponses tend to reflect the longer-term (hours-to-days) responses. This control
172 R.K. Monson

framework is embedded within two metabolic pathways, the mevalonic acid (MVA)
pathway in the cytosol and the methyl-erythritol phosphate (MEP) pathway in
plastids. Recent studies have revealed that there is certain cross-talk between these
pathways, although not always very strong. The cross-talk is coordinated to control
the use of common substrates in the synthesis of BVOCs. The MEP pathway,
which produces isoprene and the light-dependent monoterpenes, may be subject to
feedback control from its end-product dimethylallyl diphosphate (DMADP), though
the exact nature of this control is still uncertain.
The emissions of oxygenated BVOCs show evidence of control by substrate
channeling between glycolysis and mitochondrial respiration in the case of emitted
bursts of acetaldehyde, whereas the emissions of green leaf volatiles (mostly C5 and
C6 aldehydes) are due to oxidation of fatty acid chains in leaf lipids, triggered by
mechanical injury. Methanol is emitted in large quantities from expanding leaves,
and appears to be the product of pectin demethylation during cell wall expansion.
Methanol emissions are largely under stomatal control in the shorter term, but under
phenological control over leaf development in the longer term.
Many of the studies conducted to date have concerned controls over one type
of BVOC or another, but an integrated perspective as to how cells regulate the
biosynthesis and emission of an entire suite of BVOCs, many of which are likely to
carry out similar functions, has not been achieved. Interactions among the substrates
exchanged between pathways, and coupled responses to cellular cues are likely
to create a level of complexity that we have not even begun to appreciate in the
ultimate control over BVOC emissions. The emergence of new tools within the
realm of genetic manipulation, and the metabolic variants that are produced by such
manipulation, are likely to create new opportunities to ‘unpeel’ the layers of control
that regulate this complexity in control dynamics.

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Chapter 7
The Roles of Stomatal Conductance
and Compound Volatility in Controlling
the Emission of Volatile Organic Compounds
from Leaves

Peter C. Harley

Abstract Plants emit more than 30,000 different volatile organic compounds and
the extent to which stomata exert control over the emissions of these compounds
varies widely. Each of these compounds has unique physico-chemical characteris-
tics, including volatility that characterizes the partitioning between water/air and
water/lipid phases. For different volatile compounds, volatility differs by over six
orders of magnitude. The volatility of each compound, and to a lesser extent the
anatomical characteristics of the leaf, determine for each compound the extent to
which the aqueous and lipid phases within the leaf comprise temporary non-specific
storage pools between the site of synthesis and the substomatal cavities. This chapter
emphasizes that the pool size of each volatile in leaf lipid and water phases is the
chief determinant of the strength of stomatal control as well as the responsiveness
of emissions to rapid changes in light and temperature.

7.1 Introduction

Plants emit more than 30,000 different volatile organic compounds, each with its
own unique physico-chemical characteristics. Biogenic volatile organic compounds
(BVOCs) are derived from a variety of biochemical pathways and production occurs
in various leaf compartments, including plastids, cytosol, and in cell walls. Some
volatiles are emitted more or less immediately upon their production, while others
are sequestered in specialized storage structures within, e.g., resin ducts in conifers,
or on the surface of leaves, e.g., leaf glandular hairs in the mint (Mentha) family

P.C. Harley ()

Biosphere-Atmosphere Interactions Group, Atmospheric Chemistry Division, Earth System
Laboratory, National Center for Atmospheric Research, 3090 Center Green Drive,
Boulder, CO 80301, USA
e-mail: [email protected]

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 181
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 7,
© Springer ScienceCBusiness Media Dordrecht 2013
182 P.C. Harley

(Monson 2013 in this volume). With the exception of those terpenoids that are
stored in leaf surface structures such as glandular hairs, and presumably escape
directly to the atmosphere, BVOCs are assumed to accumulate in the intercellular
air space of the leaf and exit almost entirely through the leaf stomata. Though
there may be some transfer of BVOCs across the leaf cuticle, cuticular resistance
is typically two orders of magnitude higher than stomatal resistance (Nobel 2009).
Although BVOCs may diffuse slowly through the cuticle and this flux may comprise
a significant percentage of the total flux when stomata are closed, flux across the
cuticle will be ignored in the following discussion.
In extant models of BVOC emission from leaves (e.g., Guenther et al. 2006,
2012; Monson et al. 2012; Grote et al. 2013; Guenther 2013 in this volume), short-
term control over emissions is largely relegated to leaf temperature and, for those
BVOCs whose production has been shown to exhibit a light dependency, also to
incident photosynthetic photon flux density (PPFD). In addition, production of
at least some BVOC species is directly affected by CO2 partial pressure (Loreto
et al. 2001; Rosenstiel et al. 2003). Although stomata clearly exert control over
the rate at which water escapes the leaf, and have considerable influence on CO2
flux into the leaf, current widely used models of BVOC emissions assume that the
stomata have no influence on the flux (e.g., Guenther et al. 2006, 2012). Thus,
there is the implicit assumption that the emission rates of those BVOCs which
are not stored in specialized structures are equal to the rate of production, while
for compounds that are stored, the assumption is that the emission rate equals
the rate of release from internal storage structures. This chapter examines the
validity of these assumptions, and the implications for modelling BVOC emissions.
This chapter emphasizes that for many important plant volatiles, we need to
consider not only their rate of synthesis but also their physico-chemical properties
in order to predict how the emissions respond to short-term changes in stomatal
conductance.

7.2 Theory and Experimental Evidence of Stomatal

Control of Trace Gases

7.2.1 The Theory Behind Stomatal Control of BVOC

Emissions

Those, who have been involved in attempts to model transpiration and photosyn-
thesis at the scale of the leaf, have long understood the important role of stomatal
conductance in controlling the flux of water out of, and CO2 into, the leaf. Any
gas diffusing into or out of a leaf obeys Fick’s first law, which states that the
flux is proportional to the concentration difference between the leaf intercellular
air space and the air outside the leaf boundary layer, and inversely proportional to
the sum of the aggregate resistances between them. In the absence of significant
7 The Roles of Stomatal Conductance and Compound Volatility. . . 183

flux across the leaf cuticle, and assuming a low resistance across the leaf boundary
layer, a reasonable assumption at moderate wind speeds, this resistance pathway
is dominated by the stomata. This relationship has been intensively studied for
both CO2 and H2 O vapour and the reader is referred to the excellent discussion
in Nobel (2009). Cowan (1977) introduced the measure of resistance (r) in common
usage today, in which r is expressed in molar units (m2 s mol1 ) and the most
commonly used formulation for water vapour flux (Cowan 1977; Farquhar et al.
1978) becomes,

JH2 O D .wl;H2 O  wa;H2 O / =rs;H2 O D .wl;H2 O  wa;H2 O / gs;H2 O (7.1)

where JH2 O represents the water vapour flux (transpiration rate) in mol m2 s1 ,
wl;H2 O and wa;H2 O are the mole fractions of water vapour (mol mol1 ) in the
intercellular air space of the leaf and in the ambient air, respectively, and gs;H2 O ,
the stomatal conductance to water vapour (mol m2 s1 ), is the inverse of rs;H2 O , the
stomatal resistance to water vapour.
From Eq. 7.1, it is clear that any increase or decrease in stomatal conductance
(gs;CO2 ) must lead to a corresponding increase or decrease in the flux (JH2 O ) unless
the driving force (wl;H2 O  wa;H2 O ) decreases or increases in order to counteract the
change. Whether or not the emission flux of a given trace gas species (Jx ) is under
stomatal control thus depends largely on whether or not the driving force can change
enough to compensate for any change in conductance. Since emissions from the leaf
are never sufficiently great to significantly alter the trace gas mole fraction outside
the leaf, wa,x , the question reduces to whether the intercellular partial pressure of
the compound, wl,x can increase sufficiently to balance a decrease in the stomatal
conductance to given compound, gs,x . Within this context, it becomes clear why
transpiration is under tight stomatal control (Sharkey 1991). Air inside the leaf is
assumed to be saturated with water; thus, at a given leaf temperature, both wl;H2 O
and the driving force (wl;H2 O  wa;H2 O ) are constant and any change in gs;CO2 must
result in a linear change in the flux, JH2 O .
The case for CO2 is more complex because photosynthetic rate, i.e., the flux, and
wl;CO2 , the intercellular CO2 mole fraction, are not independent, because the rate
of CO2 uptake by the enzyme Rubisco is a function of wl;CO2 . Thus, the driving
force, or CO2 partial pressure difference between the intercellular air space and
the ambient air does not remain constant as conductance changes, but decreases
somewhat with increasing stomatal conductance to CO2 , gs;CO2 (Sharkey 1991).
As a result, photosynthesis is under only partial stomatal control, increasing with
increasing gs;CO2 , but in a non-linear fashion with the extent of change depending
on wl;CO2 .
For other leaf uptake processes, the extent to which wl,x can change to com-
pensate for changes in gs,x is also limited and some degree of stomatal control
is expected. Since wl can never drop below zero, the driving force (wa;x  wl;x )
can never exceed the ambient concentration. In the case of O3 , for example, Laisk
et al. (1989) reported that intercellular ozone concentration (wl;O3 ) was near zero,
although this was probably a slight underestimation as a result of a faulty O3
184 P.C. Harley

analyzer (A. Laisk and H. Moldau, personal communication). A small positive

value of wl;O3 has been reported by Moldau and Bichele (2002). Nevertheless, wl;O3
has widely been assumed to be close to zero, since O3 is known to react quickly
with cellular components doing considerable damage along the way (Wesely 1989).
When internal ozone concentration is essentially zero, the driving force remains
constant and O3 uptake is under total stomatal control. As a result, plants can reduce
exposure to O3 damage by partially closing stomata and thereby limiting uptake. As
we will see below, in contrast to the case of deposition, for BVOCs being produced
in the leaf and emitted through the stomata, the situation is quite different, and wl
and thus the driving force (wl – wa ) can vary over a very wide range, potentially
overcoming stomatal limitations.

7.2.2 Evidence for the Lack of Stomatal Control over

BVOC Emissions

I think it is fair to say that most plant ecophysiologists, comfortable with the
analogy of CO2 and H2 O vapour exchange, were predisposed to accept a role for
stomata in limiting/controlling BVOC emissions. As early as in 1981, Tingey et
al. showed that isoprene is emitted through the stomata, by demonstrating that
emission from the hypostomatous leaves of live oak (Quercus virginiana) occurred
overwhelmingly from the adaxial (lower) leaf surface. Paradoxically though, they
also found that when stomatal conductance declined by 90 % due to water stress,
isoprene emission was unaffected. In an early attempt to understand environmental
controls over isoprene emission, Monson and Fall (1989) also clearly demonstrated
that allowing gs;H2 O to vary over a wide range, whether by varying humidity or by
the addition of abscisic acid (ABA) which leads to stomatal closure, had little or
no effect on isoprene emissions. In a follow up study (Fall and Monson 1992), this
apparent paradox was resolved in a straightforward application of Fick’s law.
In principle, Eq. 7.1 applies to any trace gas. Stomatal conductance for a given
gas (gs,x) can be directly related to stomatal conductance for water vapour by the
ratio of their diffusivities (D), i.e., gs;x D gs;H2 O .Dx =DH2 O / where the subscript (x)
refers to the trace gas of interest. Thus, if one knows its diffusivity, one can estimate
the concentration of a trace gas in the intercellular air space necessary to sustain a
given measured flux at a certain value of gs;H2 O . Fall and Monson (1992) utilised this
concept to explain the apparent lack of sensitivity of isoprene emission to changes
in stomatal conductance. Although at that time, the diffusion coefficient of isoprene
was unknown, they assumed a value of 1105 m2 s1 , which is surprisingly close to
the current experimental estimate of ca. 0.9105 m2 s1 (Niinemets and Reichstein
2003a, 2003b). Employing Eq. 7.1 for isoprene, they reasoned that if Jisop and wa,isop
remain constant, while gs,isop decreases, the only way to balance the equation was for
wl,isop to increase in direct proportion to the decrease in gs,isop . If isoprene production
inside the leaf is unaffected by the concentration of isoprene in the intercellular
air space (i.e., there is no feedback inhibition on isoprene production), then wl,isop
7 The Roles of Stomatal Conductance and Compound Volatility. . . 185

Fig. 7.1 Response of

isoprene emission, net a
photosynthesis (Pnet ) and
stomatal conductance (gs;H2 O )
to withholding of water from
the cut stem of an aspen
(Populus tremuloides) leaf
(a). Water was withheld at
time t D 17 min (arrow).
Panel (b) illustrates the
intercellular isoprene partial
pressure (wl,isop ) necessary to
sustain the fluxes shown in
(a), calculated using Eq. 7.1. b
Leaf temperature was 30 ı C
(Modified from Fall and
Monson 1992)

must rise as gs,isop declines, and a new steady-state condition will arise in which the
decline in gs,isop is exactly compensated for by an increase in wl,isop . If the rise in
wl,isop is sufficiently rapid and exactly balances the decrease in gs,isop , the flux will
remain unaffected.
Thus, when water was withheld from the cut stem of an aspen leaf (Populus
tremuloides), gs;H2 O declined from about 300 mmol m2 s1 to about 10, while
calculated wl,isop increased from about 1 to over 30 mol mol1 , but isoprene
flux was unchanged (Fig. 7.1). Fall and Monson (1992) were able to verify
experimentally that wl,isop did in fact increase dramatically with stomatal closure.
They measured the isoprene content of white oak (Quercus alba) and aspen leaves
by vacuum extraction before and after applying ABA to induce stomatal closure,
and found a 25–40-fold increase following stomatal closure in both species.
A similar lack of stomatal control over emissions of ’-pinene from Quercus ilex
was documented by Loreto et al. (1996b) who showed that, by changing ambient
CO2 concentration or air humidity, gs;H2 O could be varied over a fairly wide range
with no apparent impact on ’-pinene emissions. These isoprene and ’-pinene results
gave rise to the general notion that, given the absence of feedback inhibition,
trace gas emissions ought not to be under stomatal regulation, as any change in
conductance for given trace gas will be compensated for by a near simultaneous
increase in wl,x (Sharkey 1991; Kesselmeier and Staudt 1999). The implication was
that, for those BVOCs not stored in specialized leaf storage structures, the rate of
emission reflected only the rate of production. Similarly, for those compounds that
were known to be sequestered in specialized internal storage structures such as resin
186 P.C. Harley

ducts in conifers, the measured emission rate was assumed to reflect the rate at
which they were released from the ducts. With this theoretical underpinning, the
early BVOC emission modelling efforts incorporated no direct effects of stomata
on emissions, and this has carried over to contemporary models such as MEGAN
(Guenther et al. 2006, 2012).

7.2.3 Evidence for Stomatal Control over BVOC Emissions

As an increasing number of BVOC emission patterns were investigated and

measurements accumulated, a few reports began to cast doubt on the generalization
that stomata exerted no control over emissions. As described below, a correlation
was occasionally observed between emissions and stomatal conductance. However,
these studies also acknowledged that caution must be exercised before equating
correlation and causality. Since gs;H2 O responds strongly to varying PPFD, the
emissions of any BVOC whose production is dependent on light may correlate with
changes in gs;H2 O without the stomata actually exerting any control. For instance,
Kesselmeier et al. (1996) implied a potential role for the stomata in controlling
monoterpene emissions from Q. ilex, although they suggested that light exerted
control over both monoterpene production and stomatal aperture, and assigning
control to one or the other was ambiguous. If correlations between the emission flux
of a given trace gas species and stomatal conductance are often difficult to interpret,
one way to clearly demonstrate a level of stomatal control is to observe emissions
following stomatal closure induced by ABA or the withholding of water, where light
and temperature are held constant (again assuming that the rate of BVOC synthesis
is unaffected).
MacDonald and Fall (1993) reported the first evidence of emissions of methanol
from plants, and clearly showed a strong correlation of those emissions with
stomatal conductance in leaves of both bean (Phaseolus vulgaris) and sweetgum
(Liquidambar styraciflua). These observations were confirmed and expanded upon
by Nemecek-Marshall et al. (1995) who observed large decreases in methanol
emissions when stomatal closure was induced, either by withdrawing water from
the cut petiole of a bean leaf or by replacing water with an ABA solution (Fig. 7.2a).
In addition, following extended periods of darkness, during which the stomata
were tightly closed, large transient bursts of methanol emissions were seen upon
illumination and stomatal opening (Fig. 7.2b). Although the authors suggested
these bursts might be associated with leaf damage or the volatilization of methanol
condensed on the leaf surface, the phenomenon was better explained by postulating
the accumulation of large pools of methanol somewhere within the leaf when
stomata were closed, and their subsequent emptying when stomatal constraints were
eliminated (Niinemets and Reichstein 2003a).
In the case of organic acids, Kesselmeier et al. (1997) reported a light dependence
for emissions of formic and acetic acids, but were unable to establish whether the
7 The Roles of Stomatal Conductance and Compound Volatility. . . 187

Fig. 7.2 Relationships between rapid changes in stomatal conductance and methanol emission in
leaves of a common bean (Phaseolus vulgaris). In (a), a young stem was cut and immersed in
water. After approximately 40 min, 20 M ABA was added to the transpiration stream (arrow). In
(b), the plant had been in the dark overnight, and light (PPFD D 500 mol m2 s1 ) was turned
on at time t D 0 min. Leaf temperature was 30 ı C (Modified from Nemecek-Marshall et al. 1995)

control was direct, through production, or indirect, through changes in stomatal

conductance. Gabriel et al. (1999) also observed a strong correlation between the
emissions of formic and acetic acid and gs;H2 O . They established a definitive role
for the stomata by demonstrating that when stomatal conductance fell by over
90 % after application of ABA, the emissions of both acids were reduced by
over 50 %.
In contrast to the somewhat ambiguous data with regard to stomatal control
over monoterpene emissions mentioned above, Niinemets et al. (2002) measured
emissions of four different monoterpenes from Pinus pinea during the summer
drought when stomatal conductance experienced ‘midday depression’, falling by
75 % between 1000 and 1400 h before recovering slightly in the late afternoon. Two
of the monoterpenes, limonene and “-ocimene, were unaffected by the decreased
conductance (Fig. 7.3c), while two oxygenated species, linalool and 1,8-cineole,
clearly tracked gs;H2 O as it fell and subsequently recovered (Fig. 7.3b).
188 P.C. Harley

μ
b
g

g
c

Fig. 7.3 Changes in stomatal conductance (gs;H2 O ) and the emissions of four BVOCs from needles
of Pinus pinea in response to daily variations in leaf temperature and incident photosynthetic pho-
ton flux density (PPFD). Panel (a) depicts leaf temperature and PPFD, (b) stomatal conductance
and emissions of oxygenated monoterpenes linalool and 1,8-cineole, and (c) the emissions of non-
oxygenated monoterpenes trans-“-ocimene and limonene (Modified from Niinemets et al. 2002)

7.3 Dynamic Models Explaining Stomatal

Controls of Emissions

7.3.1 Evidence for Temporary Storage Pools for BVOCs

within Leaves

As evidence accumulated that BVOC emissions were not always closely coupled
to their assumed rate of production, and that for some compounds, changes in
stomatal conductance clearly influenced emissions, the notion arose that there
must be temporary storage pools within the leaf. Several lines of evidence pointed
in this direction (see Niinemets and Reichstein (2002) and Grote and Niinemets
(2008) for a more complete discussion). For example, the large bursts of methanol
7 The Roles of Stomatal Conductance and Compound Volatility. . . 189

emission upon re-illumination following extended periods of darkness (Fig. 7.2b)

were interpreted to reflect storage within the leaf, the pools emptying rapidly upon
stomatal opening (Niinemets and Reichstein 2003a, b; Hüve et al. 2007; Harley et al.
2007). Similarly, although emissions of light-dependent monoterpenes in Quercus
ilex fall rapidly upon leaf darkening, Loreto et al. (1996a) observed that they did not
drop immediately to zero despite the fact that production was assumed to cease.
Indeed, after a sharp drop in the first few minutes, emissions continued to fall
slowly but were still measurable over an hour later, suggesting the slow emptying
of temporary storage pools. Similar results were reported by Pio et al. (2005) and
Schuh et al. (1997). In an experiment using a non-emitting chemotype of Quercus
suber, Delfine et al. (2000) found that fumigating with a mixture of monoterpenes
lead to leaf uptake, followed by monoterpene emissions that were measurable
over 12 h after fumigation ceased. This clearly indicated that monoterpenes can
accumulate in storage pools within the leaf despite the absence of specific storage
structures. This was confirmed by direct measurement of monoterpene pools in
leaves of Q. ilex (Loreto et al. 1998). Although these pools were small relative to
the pools in species with specialized storage structures, the pools were sufficiently
large to sustain steady-state emissions for approximately 15 min in darkness, a result
consistent with the leaf darkening experiment referred to above. Loreto et al. (1998)
suggested that these temporary storage pools were related to BVOC solubility and
the partitioning of BVOC between the gas and liquid phases.
At physiologically relevant ambient air concentrations, Copolovici et al. (2005)
observed significant uptake of the hydrophobic monoterpene ’-pinene and hy-
drophilic monoterpene ’-terpineol using a proton-transfer reaction mass spec-
trometer (PTR MS). Surprisingly, monoterpene uptake did not saturate with time,
suggesting that in addition to accumulating in temporary storage pools, monoter-
penes might have been metabolized inside the cell. Noe et al. (2008) found
that limonene was taken up and re-emitted from a variety of plants and saw a
strong correlation between uptake rates and plant lipid content, suggesting that
hydrophobic BVOCs might be temporarily stored in the lipid phase of the leaf.

7.3.2 Role of Temporary Storage in Stomatal

Sensitivity of Emissions

As we have seen, any change in stomatal conductance must lead to a transient

change in the intercellular partial pressure of BVOC. A new steady-state will
eventually be achieved in which the newly achieved partial pressure exactly
compensates for the change in gs and the flux returns to its value prior to the stomatal
adjustment. In the steady state, therefore, stomata can exert no control over BVOC
emission. The existing evidence summarized here, however, suggests the existence
of non-specific and temporary BVOC storage pools within all leaves. If these storage
pools are very small, as for isoprene or ’-pinene, the readjustment is very rapid and
any change in the emission rate is almost undetectable, implying that the emission
190 P.C. Harley

rate closely reflects the rate of production. If, on the other hand, these storage pools
are large and slow to re-equilibrate, the time required to return to the steady state
may be substantial, and until new steady-state conditions are obtained, emissions
will be subject to stomatal control. What then is the nature of these non-specific and
temporary storage pools?
These transient storage pools arise because all BVOCs, regardless of where in
the leaf they are produced (plastids, cytosol, in association with cell membranes)
or stored (resin ducts), must diffuse through aqueous phases in the mesophyll, a
complex series of lipid bilayer membranes, and internal air spaces before they are
emitted to the atmosphere via the stomata. Along this complex diffusion pathway,
BVOCs partition, to a greater or lesser extent, in the liquid and lipid phases,
depending on the physico-chemical characteristics of the given BVOC species and
on the amount of liquid water and lipids within the leaf (Niinemets and Reichstein
2003a, b).
To explain the various responses of different BVOCs to stomatal conductance
(and ignoring for the moment the potential role of the leaf lipid phase), Niinemets
et al. (2002) proposed that the aqueous phase of the leaf constituted a temporary
storage pool for highly soluble BVOCs, and that the susceptibility of a chemical
species to stomatal regulation was directly related to its Henry’s law constant
(H, Pa m3 mol1 ) which determines the partitioning of a volatile between the liquid
and the vapour phases. Table 7.1 lists Henry’s law constants for a wide range of
BVOCs commonly found in leaf emissions.
For isoprene (H D 7,780 Pa m3 mol1 at 25 ı C) and ’-pinene (H D 13,600 Pa m3
mol1 ) that are very insoluble in the aqueous phase of the leaf, the aqueous storage
pool is very small and the newly produced compounds partition almost entirely
to the gas phase. As soon as a new molecule is produced, it can diffuse from
the site of production to the intercellular air space in the substomatal cavity. This
would also apply to limonene (H D 2,805 Pa m3 mol1 ) and trans-“-ocimene
(H D 3,330 Pa m3 mol1 ) in Fig. 7.3c. For highly soluble compounds (Table 7.1
for Henry’s law constants), such as alcohols, e.g., methanol, 2-methyl-3-buten-2-ol,
and carbonyls, e.g., carboxylic acids (formic acid and acetic acid), and aldehydes
(formaldehyde and acetaldehyde), as well as for oxygenated monoterpenes such as
linalool and 1,8-cineole in Fig. 7.3b, newly produced compounds partition strongly
to the aqueous phase and large increases in the aqueous phase concentration are
necessary to support increases in gas-phase partial pressure sufficient to re-establish
steady-state emissions. In effect, dissolution in the aqueous phase constitutes a
temporary storage pool. Eventually, continuing BVOC production will lead to
aqueous phase concentrations sufficient to support large enough increases in the
gas phase concentration to establish a new equilibrium. Steady-state conditions
will return and emission rate will once again equal the rate of production. In such
circumstances, stomata will exert some level of control over the emissions until a
new steady state is reached. Thus, it is the time required to achieve a new steady-
state following a perturbation (e.g., stomatal closure, change in production rate)
that determines whether or not the emissions of a given BVOC are under stomatal
control.
7 The Roles of Stomatal Conductance and Compound Volatility. . . 191

Table 7.1 Henry’s law constants (gas/liquid phase partition coefficient)

and octanol/water partition coefficients of selected BVOCs at 25 ı C
Henry’s law constant Octanol/water partition
Compound (Pa m3 mol1 ) coefficient (mol mol1 )
Alcohols
Ethanol 0.507 0.490
Methanol 0.461 0.170
Aldehydes
Acetaldehyde 7.00 0.457
Formaldehyde 0.0305 0.457
Ketones
Acetone 3.88 0.575
Organic acids
Acetic acid 0.0133a 1.74a
Formic acid 0.0176a 0.288a
Terpenoids
Camphene 1,600 28,510
3-Carene 13,640 40,740
p-Cymene 1,100 31,620
Isoprene 7,780 263
Limonene 2,850 30,550
Myrcene 6,300 21,630
cis-“-Ocimene 2,470 23,530
trans-“-Ocimene 3,330 28,200
’-Phellandrene 6,950 38,460
“-Phellandrene 5,670 38,160
’-Pinene 13,600 30,900
“-Pinene 6,830 26,300
Sabinene 6,450 42,660
’-Terpinene 1,960 5,060
”-Terpinene 3,590 31,620
’-Terpinolene 2,600 29,510
Oxygenated terpenoids
Bornyl-acetate 44.3 7,244
Camphor 1.22 219
1,8-Cineole 13.6 403
Linalool 2.09 933
Menthol 1.54 2,300
2-Methyl-3-buten-2-ol 1.56 17.8
’-Terpineol 0.239 955
Thymol 0.12 1,995
Adapted from Niinemets and Reichstein (2002) and Niinemets and
Reichstein (2003a) where the original references may be found
a
All physico-chemical properties apply to undissociated form only
192 P.C. Harley

Fig. 7.4 Illustration of the decay in monoterpene emission rate following leaf darkening at time
t D 0 in holm oak (Quercus ilex) (data of Loreto et al. 1996a). The data were fit by both a single-
exponential model (the emission flux, J D J0 ekt ) where J0 is the initial emission rate and k is the
decay rate constant, and a double-exponential model (J D J0 [˜ek1t C (1˜)ek2t ]) where k1 and
k2 are the rate constants of the two pools and ˜ is the initial fraction of monoterpenes emitted from
the ‘fast’ pool (Modified from Niinemets and Reichstein 2002)

The leaf aqueous phase, thus, constitutes one temporary storage pool for water-
soluble BVOCs. Niinemets and Reichstein (2002) examined more closely the
relatively slow decline in light-dependent monoterpene emissions following dark-
ening in Q. ilex (Loreto et al. 1996a) and observed that the decay kinetics were not
well described by an exponential decay model, as would be expected if a single pool
were being emptied. The data was well described however by a double-exponential
model, which implicitly assumes the presence of two independent temporary storage
pools, a ‘fast’ pool and a ‘slow’ pool, emptying at different rates (Fig. 7.4).
The ‘fast’ pool was shown to be the aqueous-phase pool described above, which
empties in a few seconds, consistent with the discussion above, assuming vales of
H greater than 500 Pa m3 mol1 . Consistent with the observations of Noe et al.
(2008), the ‘slow’ pool was assumed to reside in the lipid phase of the leaf in
which hydrophobic but lipid-soluble BVOCs might temporarily reside. Thus, in a
manner analogous to the aqueous phase, temporary storage in the lipid phase can
result in non-steady-state conditions under which the stomata might exert some
control over emissions. The extent to which a given BVOC partitions into the
lipid phase is related to its octanol/water partition coefficient (Ko/w , mol mol1 ),
defined as the ratio of the solubility of a compound in octanol (a non-polar solvent)
to its solubility in water (a polar solvent). Values of Ko/w for BVOCs of interest
are given in Table 7.1. The higher the Ko/w , the more non-polar the compound
and the more likely it is to partition to the non-polar lipid phase. BVOCs with
low values of H (highly water-soluble) will thus tend to have low values of Ko/w
7 The Roles of Stomatal Conductance and Compound Volatility. . . 193

Fig. 7.5 Relationship

between Henry’s law constant
(H) and octanol/water
partition coefficient (Ko/w ) for
31 commonly emitted
BVOCs (Table 7.1).
Unsubstituted hydrocarbons
are differentiated from those
containing at least one
oxygen atom

(low lipid solubility) and vice versa (Fig. 7.5). A detailed discussion of both Henry’s
law constant and octanol/water partition coefficients, including estimates of their
temperature dependencies, is found in Copolovici and Niinemets (2005).

7.4 A Dynamic (Non-Steady-State) Model of BVOC

Emissions

7.4.1 Description of Tentative BVOC Pools

and Their Dynamics

As discussed above, evidence has accumulated that both the aqueous and lipid
phases of the leaf can act as non-specific, temporary storage pools. The recognition
that the water solubility of a gas, and to a lesser extent the lipid solubility, largely
determine whether or not it came under stomatal regulation, lead to the development
of a dynamic BVOC emission model that resolved the apparently contradictory
reports in the literature regarding the stomatal regulation of trace gas emissions.
Building on the concepts outlined above, Niinemets and co-workers (Niinemets
et al. 2002; Niinemets and Reichstein 2002, 2003a, b; Noe et al. 2006) developed
a dynamic model of BVOC emissions, depicted conceptually in Fig. 7.6, which
incorporates these ideas and forms the basis for the remainder of this chapter.
Upon its production (or release from permanent storage structures) at a rate I
(mol m2 s1 ), each BVOC partitions into the aqueous and lipid phases of the
leaf mesophyll, the partitioning ratio being determined by an empirical partitioning
coefficient, . These non-specific, temporary storage pools (mol m2 ) are designated
Sa (aqueous) and Sl (lipid). The rate of release of each BVOC from the aqueous
storage pool (Sa ) is described by a compound-specific first-order kinetic constant,
194 P.C. Harley

Fig. 7.6 Schematic diagram Rate of synthesis

of the dynamic model of (or release from pool)
Niinemets and Reichstein I
(2002, 2003a, b), illustrating
the potential role of h 1- h
non-specific storage pools in
controlling BVOC emissions.
BVOCs are produced Sa Sl
(or released from storage Aqueous phase Lipid phase
structures) at a rate I and
eventually diffuse through the
stomata to the ambient air at
Mesophyll ka kl
rate J. Along this complex
diffusion pathway, BVOCs
may be stored temporarily in
Intercellular air space
Sg
three non-specific storage Gas phase
pools – an aqueous-phase (Sa ) (sub stomatal cavity)
or lipid-phase (Sl ) pool in the gias
mesophyll or a gas-phase kg
pool (Sg ) in the leaf
intercellular air space. Further Ambient air gs
details in the text (Modified
from Noe et al. 2006)
J
Emission rate

ka (s1 ), that is related both to the complex diffusion pathway within the leaf and
to the physico-chemical characteristics of the compound, especially its Henry’s law
constant and diffusion constant in liquid phase. Analogously, release from the lipid
storage pool (Sl ) is related to kl (s1 ), dependent again on the diffusion path, as well
as on the octanol/water partition coefficient of the compound. These time constants
describe the dynamics of BVOC release from Sa and Sl into the leaf gas phase (Sg ,
mol m2 ) in the leaf intercellular air space. The flux out of the leaf gas-phase pool
and into the ambient air, i.e., what we typically measure, is described by a third
rate constant, kg (s1 ) that is determined by the sum of the gas phase conductance
from the outer surface of cell walls to the substomatal cavity (intercellular air
space conductance, gias , mol m2 s1 ) and the stomatal conductance between the
intercellular air space and the ambient air (gs , mol m2 s1 ).
For a complete derivation of the equations used in the dynamic emissions model,
the reader is referred to the original model description describing the role of the
aqueous-phase pool (Niinemets et al. 2002; Niinemets and Reichstein 2003a, b)
and the expanded version incorporating non-specific lipid phase storage pools as
well (Niinemets and Reichstein 2002; Noe et al. 2006). In general, the dynamics
of the aqueous-, lipid- and gas-phase pools are described by a system of ordinary
differential equations:

d Sa .t/
D I  ka Sa .t/ (7.2)
dt
7 The Roles of Stomatal Conductance and Compound Volatility. . . 195

d Sl .t/
D .1  /I  kl Sl .t/ (7.3)
dt
d Sg .t/
D ka Sa .t/ C kl Sl .t/  kg Sg .t/ (7.4)
dt
where the measured emission rate of the volatile from the leaf (J) is

J D kg Sg .t/ (7.5)

The kinetic constants ka, kl and kg are all related to leaf structural properties defining
the size of the aqueous-, lipid- and gas-phase pools within the leaf, the relevant
transfer conductances between the pools, and, most importantly, the physico-
chemical characteristics of the BVOCs (H and Ko/w ). All the kinetic constants
require an estimate of the ratio of leaf area (A, m2 ) to leaf volume (V, m3 ).
Additionally, the fractions of the total leaf volume (m3 m3 ) comprising air spaces
(fias ), liquid water (fw ) and lipids (flip ) are needed for the determination of kg ,
ka and kl , respectively. In addition, estimates of the total gas phase, liquid phase
and lipid phase diffusive conductances from the site of production (or release
from specialized storage pools) are required for each BVOC of interest. These
conductances depend on diffusion pathway length, and compound diffusivities in
given phases.

7.4.2 Sensitivity of the Model to Pool Sizes as Determined

by Leaf Structure and Compound Solubility

The rate constant of the gas-phase, kg , is related to the size of the gas-phase pool and
to the total gas phase diffusion conductance (GG , mol m2 s1 ) between the outer
surface of the mesophyll cell walls and the ambient air:

A RT
kg D GG (7.6)
V fias P

where R is the gas constant (8.314 Pa m3 mol1 K1 ) and RT/P converts conduc-
tance to units of m s1 . GG represents the sum of the stomatal conductance for the
BVOC in question and the internal conductance from the mesophyll cell walls to
the substomatal cavity Gias . Given the serial conductances, GG D 1/(1/Gs C1/Gias ).
Using Eq. 7.6, one can calculate a range of values for kg , which scales linearly with
both A/(V fias ) and GG . The half-time (s) for gas pool turnover ( g D ln(2)/kg ) then
decreases logarithmically as GG increases. Plotting £g as a function of GG (Fig. 7.7)
for two values of Vfias /A [the volume of air inside the leaf per unit leaf area (m3 air
m2 leaf)], representing the likely range of values for a variety of leaf morphologies,
several important things become apparent.
196 P.C. Harley

Fig. 7.7 The half-time

required for turnover of the
gas-phase pool ( g ) as a
function of stomatal
conductance, assuming two
different values for the
amount of air within the leaf

τ
(cm3 m2 ). This example
assumes a diffusivity similar
to that of ’-pinene (diffusion
coefficient of 6106 m2 s1
at 25 ı C)

The gas pool turnover time increases as stomatal conductance decreases, and
as the total volume of air inside the leaf increases. However, the turnover of the
gas pool is very rapid, on the order of a few seconds, except at very low values
of conductance. Thus, it is assumed that the gas pool reaches a new steady-state
value very rapidly following a perturbation, much more rapidly than stomatal
movements occur, and cannot therefore contribute significantly to any observed
stomatal limitation. Any significant delay in reaching a new steady-state value of
wl must therefore be due to the turnover time of the liquid- and/or lipid-phase pools,
determined by the parameters ka and kl , respectively.
If we consider the gas-phase to be in the steady state, ka will depend on the liquid
pool size (Vfw /A) as well as the total liquid phase conductance between the site of
synthesis and the outer surface of the cell walls (GA , m s1 ) and the gas phase
conductance, GG .

GA fwAV
ka D (7.7)
1C GA P
GG H

where P is the atmospheric pressure. This equation assumes that, following any
perturbation, the gas pool re-equilibrates extremely rapidly and may always be
considered to be in a steady state. As shown below, this assumption is almost
always valid, and contrasts with the situation for the aqueous and lipid pools, which
are frequently in a non-steady-state condition. Importantly, the value of ka is also
strongly dependent on the solubility of the BVOC in question, defined by H (see
Niinemets and Reichstein 2003a for complete derivation). In contrast to kg , which is
constrained to a relatively narrow range of values, all fairly small, the incorporation
of H, which can vary over several orders of magnitude for different BVOCs
(Table 7.1), allows ka to vary over a large range. This translates into a range of liquid
7 The Roles of Stomatal Conductance and Compound Volatility. . . 197

Fig. 7.8 The half-life of the

aqueous pool ( a ) as a
function of stomatal
conductance for a range of
Henry’s law constants
(Pa m3 mol1 ). The aqueous
phase conductance, GA , is
assumed to be 105 m s1
and the liquid pool size to be
100 cm3 m2

pool half-lives, from quite short to very long. As we have seen, the latter correspond
to highly soluble BVOCs, with low values of H and long pool turnover times that
are often in a non-steady-state condition and thus susceptible to stomatal regulation.
The aqueous phase conductance, GA , is a complex term, reflecting the diffusion
pathway through the plant mesophyll cells and across various membranes. The
length of the diffusion path and the resistances encountered differ depening on leaf
architecture and the specific site of BVOC synthesis. Estimates of the liquid phase
resistances encountered by various BVOCs in leaves of Pinus sylvestris, Phaseolus
vulgaris and Quercus ilex (Niinemets and Reichstein 2003a) are available, but
estimates of GA remain somewhat uncertain and GA is expected to differ across
plant species. Fortunately, as demonstrated by Niinemets and Reichstein (2003b),
the predicted leaf behavior is relatively insensitive to the value of GA . For the
purpose of illustration, we will use a value of 105 m s1 , typical of short-chain
aliphatic compounds (Niinemets and Reichstein 2003a), in the following analysis.
Figure 7.8 illustrates the importance of H in determining the turnover time of
the aqueous pool. As expected, the pool turns over more rapidly as gs;H2 O increases,
the half-life of the pool ( a ) decreasing by over an order of magnitude as gs;H2 O
varies from very low to high values. But the effect of H is even more dramatic,
with  a decreasing by several orders of magnitude as H varies from 0.5 to 5,000,
approximately the range observed for plant BVOCs. Note that for those BVOCs with
values of H exceeding 50 Pa m3 mol1 , the half-life of the liquid pool is less than
3 min, even at very low values of gs;H2 O . Stomatal control of emission is unlikely
to be of significance for compounds in this range, as aqueous- and gas-phase pools
will reach a new steady state following any perturbation in a few seconds to a few
minutes, depending on stomatal conductance, and emission will simply equal the
rate of production or the rate of release from the internal structural storage pools.
As noted above, such lack of stomatal control has been reported for very insoluble
compounds isoprene, ’-pinene, trans-“-ocimene and limonene (Table 7.1), and
this will be the case for all non-oxygenated terpenoids (H > 2,000 Pa m3 mol1 ,
198 P.C. Harley

Fig. 7.9 The half-life of the

aqueous pool ( a ) as a
function of stomatal
conductance for a range of
leaf water contents. The
liquid phase conductance,
GA , was assumed to be
105 m s1 , and the assumed
Henry’s law constant was
0.5 Pa m3 mol1

Table 7.1). In contrast, a variety of oxygenated BVOCs, including organic acids,

ketones, aldehydes and alcohols (including terpenoid alcohols) have H values less
than 5 Pa m3 mol1 (Table 7.1) and have been shown to be under some level of
stomatal control.
A second parameter which strongly affects the turnover time of the aqueous pool,
and therefore the extent to which stomata can limit emissions, is the size of the
aqueous-phase pool into which a given BVOC can partition. The rate constant, ka ,
is inversely proportional to the size of the pool (Eq. 7.7) which depends on both the
surface to volume ratio of the leaf and the fraction of the leaf volume occupied by
liquid water. Figure 7.9 illustrates the impact of varying the liquid pool size from 50
to 200 cm3 H2 O per m2 of leaf on the aqueous-phase pool turnover time (£a ). For
obvious reasons, a leaf containing less liquid water into which a BVOC can partition
will reach equilibrium more quickly and stomata will have less opportunity to exert
control, even for very soluble compounds.

7.4.3 Dynamic Model Applications in Explaining

the Stomatal Controls

As discussed above, among the early evidence of complex stomatal controls on

certain trace gas emissions were the observed bursts of methanol emissions when
stomata opened following a prolonged period of darkness (Nemecek-Marshall et al.
1995; Hüve et al. 2007; Harley et al. 2007). The dynamic model simulates such
behavior well. To demonstrate the stomatal controls during rapid stomatal closure
and subsequent re-opening, emissions of three hypothetical BVOC species with
values of H ranging from 0.5 to 50 Pa m3 mol1 were simulated (Fig. 7.10). These H
values encompass the range of water solubility over which changes in conductance
7 The Roles of Stomatal Conductance and Compound Volatility. . . 199

Fig. 7.10 Simulations using

the dynamic model of
Niinemets and Reichstein
(2003a) illustrating the effect
of rapid changes in stomatal
conductance (a) on the
emissions of three
hypothetical BVOCs with
different Henry’s law
constants (H, Pa m3 mol1 )
(b). The production rate of all
three is set to 0.5 nmol m2 s1
and GA to 105 m s1 . Also
shown (c) are the simulated
changes in the size of the
aqueous pool

are expected to have a significant impact on emissions. Note that prior to stomatal
closure, when the emissions are in a steady state, the liquid pool size required to
sustain emission of 0.5 nmol m2 s1 is much greater for the most soluble BVOCs
(Fig. 7.10). This reflects the circumstance that to achieve the gas phase gradient
required to sustain the flux, the liquid pool concentration must be greater. Following
a rapid change in gs;H2 O from 100 to 10, all three BVOCs experience a transient
reduction in emissions, but the pool dynamics are dramatically different. While the
least soluble gas (H D 50 Pa m3 mol1 ) reaches a new, higher, steady-state liquid
pool size within 7 min, the more soluble (H D 5 Pa m3 mol1 ) requires about an
hour (Fig. 7.10). In both cases, the BVOC emission flux has ultimately returned to
its pre-closure value. In the case of the most soluble gas (H D 0.5 Pa m3 mol1 ,
200 P.C. Harley

Fig. 7.11 Methanol a

emissions from a leaf of a g
sorghum-sudangrass hybrid
(Sorghum bicolor  S. bicolor
subsp. sudanense). The leaf
was kept in darkness for 14 h

μ
prior to the experiment, and
the light was turned on at time
t D 12 min. Leaf temperature

g
was 30 ı C until time b
t D 100 min, when it was
increased to 35 ı C for the rest
of the experiment. Stomatal
conductance (a) responded
rapidly to abrupt changes in
PPFD, accompanied by near
simultaneous changes in
methanol emissions
(b, measured). The dynamic
model predicted rapid
increases in liquid-phase c
pools of methanol upon
stomatal closure and
depletions upon stomatal
μ

opening (c), accompanied by

sudden bursts of emission
(b, predicted) (Modified from
Harley et al. 2007)

similar to methanol) the BVOC is still accumulating rapidly in the aqueous phase
after 75 min and the emission rate remains less than half of its pre-closure level.
Upon rapid stomatal opening at time t D 100 min., equilibration of the liquid
pools is greatly accelerated in all three cases, because gs;H2 O is much higher, but the
effect of H on the time required to reach the equilibrium remains apparent. When
H  50 Pa m3 mol1 , as it is for all non-oxygenated hydrocarbons, equilibration
of the liquid pools requires only a few seconds, and stomata exert no control over
emissions (not shown).
Niinemets and Reichstein (2003a, Fig. 6) demonstrated that their proposed
dynamic model simulated well the emission burst in the data of Nemecek-Marshall
et al. (1995). Figure 7.11 provides another example (modified from Harley et al.
2007) illustrating not only a large methanol burst following a prolonged period of
darkness, but also that even short periods of stomatal closure (ranging from 4 to
10 min.) are sufficient to allow the accumulation of methanol in the aqueous phase
with subsequent release upon rapid stomatal opening. A leaf of sorghum-sudangrass
hybrid (Sorghum bicolor  S. bicolor subsp. sudanense) had been in darkness for
14 h and at the end of the dark period, gs;H2 O was extremely low and emissions
7 The Roles of Stomatal Conductance and Compound Volatility. . . 201

were near zero. When the light was turned on at time t D 12 min., stomata opened
rapidly, accompanied by a large emission burst which lasted over an hour before a
new steady state was established with emissions of approximately 1.5 nmol m2 s1 ,
presumably reflecting the rate of production. Later in the day, the leaf was subjected
to three light off-light on cycles, and each time, gs;H2 O fell rapidly, as did methanol
emissions, to near zero. Each time the light was turned on, stomata re-opened and
short bursts of methanol emissions 4 to 5 times those observed prior to darkening
occurred. The size of the burst was proportional to the amount of time spent in the
dark, reflecting the expected increase in the size of the aqueous pool (bottom panel)
which accumulated under conditions of low stomatal conductance.
The data in Fig. 7.11 clearly demonstrate that stomata can exert some control
over emissions of water-soluble BVOCs under the somewhat artificial experimental
conditions of sudden and rapid stomatal closure and re-opening. Is the impact
of varying stomatal conductance still apparent under more natural experimental
conditions in which light and temperature are allowed to fluctuate, leading to
both changes in methanol production rates and more gradual changes in gs;H2 O ?
To address this, rates of methanol emission and gs;H2 O of mature needles of
loblolly pine (Pinus taeda) were measured during experimental modifications of
temperature and light (Fig. 7.12a). Methanol production (Fig. 7.12b) was assumed
to increase exponentially with increasing temperature but to be insensitive to
incident PPFD. Measured emissions departed significantly from assumed rates
of production, sometimes exceeding production, particularly when stomata were
opening, and sometimes falling below assumed production rates, especially when
gs;H2 O was declining. Emissions predicted using the dynamic model of Niinemets
and Reichstein (2003a) closely mimicked much of the observed behaviour, clearly
implicating the stomata in short-term control of methanol emissions.
Although it is assumed that methanol production is insensitive to PPFD, gs and
PPFD clearly covary and it is problematic to assign control to one or the other.
However, if methanol emissions are plotted against PPFD (Fig. 7.13a) and gs;H2 O
(Fig. 7.13b), a stronger relationship with gs;H2 O is clearly indicated. As expected,
for a given value of gs;H2 O , emissions are higher when stomata are opening than vice
versa. When gs;H2 O is increasing, liquid- and gas-phase pools that had accumulated
at lower gs;H2 O are now emptying, resulting in higher emissions that exceed the
assumed rate of production (Fig. 7.12c, d).

7.4.4 Dynamic Model Applications: Role of the ‘Slow’ Pool

To this point, I have focused on the temporary non-specific storage pool represented
by the liquid fraction of the leaf, the ‘fast’ pool depicted in Fig. 7.4, and the
consequences of this pool for highly soluble BVOCs (H < 50 Pa m3 mol1 ). I
now return to the ‘slow’ pool residing in the lipid phase of the leaf, encountered
by BVOCs as they diffuse across lipid bilayer membranes. As depicted above
(Fig. 7.6) the flux from this pool is determined by the size of the lipid pool (Sl ), the
202 P.C. Harley

1200 40

Leaf temperature (°C)

PPFD (µmol m–2 s–1)
a PPFD
1000 36
800 32
600 28
400 24
200 20
0 16
100 1.2

Methanol production
b
80 1

(nmol m–2 s–1)

0.8
60
0.6
40
0.4
g
20 0.2
Production
g

0 0

1.2
c
Methanol emission

1
(nmol m–2 s–1)

0.8

0.6

0.4

0.2 Measured
Predicted
0
Aqueous pool (nmol m–2)

1 4000
d
Gas pool (nmol m–2)

0.8
3000
0.6
2000
0.4
1000
0.2

0 0
8 9 10 11 12 13 14 15 16 17
Time (hours)

Fig. 7.12 Methanol emissions from needles of loblolly pine (Pinus taeda). Incident PPFD and
leaf temperature (a) were varied and stomatal conductance (b) and methanol emissions (c) were
measured. The fit obtained using the dynamic model of Niinemets and Reichstein (2003a) is shown
in (c) and modelled rates of methanol production (b) and aqueous- and gas-phase pools (d) are also
shown (Modified from Harley et al. 2007)
7 The Roles of Stomatal Conductance and Compound Volatility. . . 203

1.2
Methanol flux (nmol m-2 s-1)
a b
1

0.8

0.6

0.4
PPFD increasing g
0.2
PPFD decreasing g
0
0 300 600 900 1200 0 20 40 60 80 100
PPFD (mmol m-2 s-1) gS,H2O (mmol m-2 s-1)

Fig. 7.13 Relationship between methanol emissions and incident PPFD (a) and stomatal conduc-
tance (b) for the data presented in Fig. 7.12. Data were divided into periods of either increasing or
decreasing conductance (Modified from Harley et al. 2007)

fraction of produced BVOCs which enters this pool (1˜) and the rate constant, kl .
Analogously to the situation with respect to ka , kl is determined by leaf structure and
diffusive properties (analysed in detail by Niinemets and Reichstein 2002) and the
solubility of a given BVOC in the lipid phase. This latter property has been shown
to be well approximated by the octanol/water partition coefficient (Ko/w , mol mol1 )
(Niinemets and Reichstein 2002; Copolovici and Niinemets 2005; Noe et al. 2006).
Thus,
 
A Pi
kl D GL 1 (7.8)
V flip Cl KA=L

where GL (m s1 ) is the diffusion conductance from the lipid storage pools to the
substomatal cavity, Cl (mol m3 ) is the lipid phase concentration, Pi (Pa) is the
partial pressure of the trace gas in the substomatal cavity, flip is the lipid volume
fraction in the leaf (m3 m3 ) and KA/L (Pa m3 mol1 ; approximated as H/Ko/w – see
Niinemets and Reichstein 2002 for details) is the air/lipid phase partition coefficient.
Thus, in addition to the leaf structural characteristics and lipid pathway diffusion
resistances, kl is related to both H and Ko/w . As shown above (Fig. 7.5), H and Ko/w
are strongly correlated. Thus, those water-soluble BVOCs with potentially large
liquid phase storage pools are unlikely to have significant transient storage pools
in the lipid phase, and vice versa.
The relative importance of temporary storage in the aqueous and lipid phase
pools for two contrasting terpenoid species, linalool, a monoterpene alcohol
(H D 2.09 Pa m3 mol1 ; Ko/w D 933 mol mol1 ) and trans-“-ocimene (H D 3,330;
Ko/w D 28,200) has been demonstrated in the Mediterranean conifer Pinus pinea
(Fig. 7.14). Following the establishment of steady-state emissions, Noe et al. (2006)
replaced 12 CO2 in the air stream with 13 CO2 at time t D 10 min., then measured
the incorporation of 13 C into the two compounds until labelling was terminated
204 P.C. Harley

Fig. 7.14 Measured

13
C-labelling and de-labelling
rates of linalool (a) and
trans-“-ocimene (b) in
needles of the Mediterranean
conifer Pinus pinea.
Fumigation with 13 CO2 began
at time t D 10 min. and ended
at time t D 40 min. Half-times
of the aqueous ( a ) and
lipid-phase ( l ) pools and the
fractional partitioning into the
aqueous pool () are as
shown for both compounds.
Also shown are the model
simulations of labeled
emissions arising from each
pool and their sum (Modified
from Noe et al. 2006)

at 40 min, after which they monitored the gradual loss of 13 C label (Fig. 7.14).
Both incorporation and subsequent loss of the label were much more rapid for
ocimene than for linalool, consistent with the vastly differing Henry’s law constants
of these compounds. Simulations obtained using the dynamic model, and assuming
two storage pools, are illustrated in the figure using pool half-lives (a and 1 ) and
the fractional partitioning coefficients () indicated (for additional information on
model parameterization, see Noe et al. 2006). For linalool (Fig. 7.14a), labelling
and de-labelling dynamics were explained almost entirely by aqueous pool storage
and storage in the lipid phase was inconsequential. For the hydrophobic ocimene,
however, changes in emission rates were slower than predicted by aqueous phase
dynamics alone. As expected, the aqueous phase equilibrated rapidly, and the
contribution of the ‘slow’ lipid-phase pool was necessary to explain the long time-
lags in both labelling and de-labelling kinetics (Fig. 7.14b). Thus, this simulation is
consistent with the inverse correlation between H and Ko/w . Temporary storage in
the lipid phase is unlikely to make a significant contribution for those water-soluble
BVOCs most susceptible to stomatal control (e.g., linalool). For relatively insoluble
BVOCs (H > 50 Pa m3 mol1 ; e.g., ocimene), however, lipid phase storage explains
continuing emissions of light-dependent monoterpenes in the dark in leaves of
Quercus ilex (Loreto et al. 1996a) and the uptake and slow release of endogenously
supplied monoterpenes (Delfine et al. 2000; Noe et al. 2008; Himanen et al. 2010).
7 The Roles of Stomatal Conductance and Compound Volatility. . . 205

7.5 Conclusions

We have seen that, in the steady state, stomatal conductance can exert no control
over emissions of a trace gas produced in the leaf. In the absence of feedback
inhibition on production, the BVOC gas phase concentration in the substomatal
cavity will increase in direct proportion to the decrease in conductance, and the
resulting increase in driving force will compensate for the increased resistance.
Whether or not stomatal limitations are observed then becomes a function of
the time required for a new steady-state condition to be achieved following any
perturbation, e.g., a change in stomatal conductance or a change in the rate of
trace gas production. The dynamic models developed by Niinemets and colleagues
clearly demonstrate that the time required varies widely between different BVOCs,
depending largely on their physico-chemical characteristics, as well as on leaf
anatomical characteristics which affect the size of the potential aqueous and lipid
phase pools and the gas phase and liquid phase resistances encountered by a gas as
it diffuses from the site of production to the substomatal cavities.
The potential impact on emissions of water-soluble BVOCs due to changes in
stomatal conductance can be dramatic, especially when stomata are induced to open
or close rapidly by, for example, suddenly turning off the light or adding ABA
to the gas stream. Under natural field conditions, with continuously fluctuating
light and temperature conditions, changes in gs are generally much more gradual,
and the impact on emissions more subtle. Nevertheless, if one is interested in
understanding the short-term controls over BVOC emissions, a dynamic model such
as that described above, is crucial. Integrated over longer time periods, however, the
importance of stomatal control becomes less evident. In Fig. 7.12, for example,
at any given time the rate of methanol emission may be significantly greater or
lower than the assumed rate of production, and can only be understood in the
context of temporary pool dynamics. Integrated over several hours or the entire day,
however, total emissions will necessarily be similar to total production since storage
in aqueous and lipid pools is temporary. An exception may be the large transient
increase in emissions observed in the morning when stomata open. For very water-
soluble compounds, such transient increases may last several hours.
What emission model to use, and whether or not stomatal effects can be
considered significant, clearly depends therefore on the nature of the question being
asked. If one is interested in understanding the short-term dynamics of BVOC
production and emission, it is critical to include stomatal effects for those BVOCs
with Henry’s law constants below approximately 50 Pa m3 mol1 (and perhaps
those with high values of Ko/w , although this is much less studied). If, on the
other hand, one is trying to predict average emissions on half-hourly or hourly
time scales for incorporation into air quality models or chemistry-transport models,
stomatal effects are less likely to strongly influence predictions, and emissions will
be influenced almost exclusively by the rate of BVOC production, in which case a
static model such as MEGAN (Guenther et al. 2006, 2012) may be appropriate.
A clear exception to this generalization is the observed mid-day depression in
206 P.C. Harley

emissions of oxygenated monoterpenes in hot, dry situations (e.g., Fig. 7.3). In such
a case, traditional emission models such as MEGAN (Guenther et al. 2006, 2012)
will greatly overestimate emissions and the dynamic model is more appropriate.

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Chapter 8
The Role of Volatile Organic Compounds
in Plant Resistance to Abiotic Stresses:
Responses and Mechanisms

Malcolm Possell and Francesco Loreto

Abstract Why plants constitutively emit certain volatile organic compounds is a

question that has attracted numerous researchers since the discovery of emissions.
A number of hypotheses exist regarding the role of constitutive volatile organic
compounds and many of these highlight the role of these compounds in enhancing
plant tolerance to certain abiotic stresses. As practically any stress can modify
constitutive emissions and also elicit production of novel compounds (induced
emissions), this chapter provides a review of the hypotheses with particular foci
on the key environmental stresses – heat and drought. Furthermore, we discuss how
changes in the atmospheric CO2 concentration over past and future geologic epochs
are likely to affect the role of volatile organic compounds as an adaptation to abiotic
stresses.

8.1 Introduction

8.1.1 Volatiles Released by Plants

A multitude of biogenic volatile organic compounds (BVOCs) are emitted by

terrestrial and aquatic ecosystems. The emitted compounds can include alkanes,
alkenes, alcohols, aldehydes, ethers, esters, carboxylic acids and a variety of
isoprenoids (terpenoids). A common feature of the diverse range of compounds
emitted is that they have a sufficiently low boiling point such that the vapour

M. Possell ()
Faculty of Agriculture and Environment, University of Sydney, Sydney, NSW 2006, Australia
e-mail: [email protected]
F. Loreto
Istituto per la Protezione delle Piante (IPP), Consiglio Nazionale delle Ricerche (CNR),
Via Madonna del Piano 10, 50019 Sesto Fiorentino, FI, Italy

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 209
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 8,
© Springer ScienceCBusiness Media Dordrecht 2013
210 M. Possell and F. Loreto

pressure at physiologically relevant temperatures is high, resulting in significant

emissions to the atmosphere. All organisms have the potential to produce BVOCs
and since the seminal work of Went (1960), the importance of terrestrial ecosystems,
and plants in particular, as sources and sinks of BVOCs has become apparent
(Kesselmeier and Staudt 1999; Fuentes et al. 2000). At least 1,700 compounds
with many isomers and derivatives are currently known to be emitted by plants
(Knudsen and Gershenzon 2006). These can be emitted from all parts of the
plants including leaves (Loreto and Schnitzler 2010), roots (e.g., Asensio et al.
2008) and flowers (Knudsen et al. 1993). BVOC production and emission can be
constitutive, in which case the emissions can be detected throughout the lifecycle
of the plant or at specific developmental stages (e.g., flowering, fruit ripening
or/and leaf maturation). Constitutive emissions respond to environmental drivers
such as light, temperature, and atmospheric CO2 concentration (Grote et al. 2013;
Li and Sharkey 2013; Monson 2013). In addition, the emission capacity varies
depending on ontogeny, nutrient availability and atmospheric CO2 concentration
during plant growth (Niinemets et al. 2010a; Monson 2013). Synthesis of specific
compounds can also be induced (induced emissions) in response to mechanical
wounding as a consequence of wind or herbivory (e.g., Laothawornkitkul et al.
2008), harvesting (Brilli et al. 2012), or even cellular damage caused by abiotic
stresses such as drought (Capitani et al. 2009), air pollution (Pinto et al. 2007;
Beauchamp et al. 2005), heat (Copolovici et al. 2012) or flooding stress (Copolovici
and Niinemets 2010).
De novo biosynthesis and emission of BVOCs includes products of the lipoxy-
genase (LOX) pathway, such as oxylipins and green leaf volatiles (GLVs; Feussner
and Wasternack 2002), a wide range of terpenoids from the mevalonate and
methylerythritol 4-phosphate pathways (homo-, mono, di-, sesquiterpenes; Lich-
tenthaler 2010), products of the shikimate pathway (e.g., methyl chavicol and
methyl salicylate) (Dudareva et al. 2006) and C1 and C2, low molecular weight,
compounds such as methanol, acetaldehyde (Kreuzwieser et al. 1999; Fall 2003;
Kreuzwieser and Rennenberg 2013; Monson 2013) and ethylene (Lin et al. 2009)
which are formed via other mechanisms. Recently, Keppler et al. (2008) identified
methane emissions from plants, under aerobic conditions, that were derived from
pectin. However, methane is not normally included in BVOC analysis as these
emissions likely reflected a “non-biogenic” response due to exposure to ultraviolet
radiation (Bruhn et al. 2012). Thus, BVOCs are often also referred to as non-
methane hydrocarbons (NMHC). BVOCs are generally classified based upon the
biosynthetic pathway they originate from. They could also be classified according to
their volatility or atmospheric lifetime to better understand their role in ecosystems
(Arneth and Niinemets 2010; Holopainen 2011).
Advances in analytical capabilities during the last 15–20 years (reviewed by
Tholl et al. 2006) have led to an increase in our knowledge of the spatial and
temporal distribution of BVOC biosynthesis and emission, and the diversity of
BVOCs produced. Consequently, for some highly studied compounds, such as iso-
prene (2-methyl-1,3-butadiene), where knowledge about the sources, biosynthesis
and emission is reasonably well constrained, different emission inventories show
8 The Role of Volatile Organic Compounds in Plant Resistance to Abiotic. . . 211

good agreement (Arneth et al. 2008; Ashworth et al. 2013; Guenther 2013). For
other compounds, such as acetone, where there is limited knowledge of sources,
biosynthesis and controls on emission, emission inventories can have large margins
of uncertainty (10–148 Tg year1 ; Singh et al. 2004; Wiedinmyer et al. 2004).
Consequently, the global flux of BVOCs from the terrestrial biosphere to the
atmosphere is highly uncertain, but is likely in the order of 700–1,000 Tg C year1
(1 Tg D 1012 g) (Guenther et al. 1995) which is comparable to the estimated global
flux of methane to the atmosphere (500–1,100 Tg year1 ; Denman et al. 2007;
Adushkin and Kudryavtsev 2010). Once emitted, BVOCs play an important role in
the oxidative capacity of the atmosphere (e.g., Fehsenfeld et al. 1992) and mediate
the formation of secondary organic aerosol (Mentel et al. 2009; Kulmala et al.
2013). BVOC fluxes can account for 5–10 % of total net carbon exchange (Peñuelas
and Llusià 2003) or about 1–2 % of the estimated global carbon assimilation
by terrestrial ecosystems (Grace and Rayment 2000). The emissions of terpenes
from plants are estimated to account for over half the total BVOC burden to the
atmosphere (Guenther et al. 1995), but model estimates greatly diverge (Arneth et al.
2008; Ashworth et al. 2013). Obviously, the production of BVOCs by vegetation,
and in particular the production of volatile isoprenoids, comes at a cost in terms of
carbon and energy and it is reasonable to assume that these costs must be balanced
by the benefits of production.

8.1.2 Plants in Natural Stressful Environments

Plants are exposed to a multitude of single and combined stresses at different

intensities and durations throughout their lifetime (Fig. 8.1). Every environmental
factor deviating from the optimum reduces the rate of primary metabolic processes,
such as those associated with reproduction and growth, thereby constituting a
stress to the plant (Niinemets 2010a). Plant physiological potentials and stress
severity determine whether the plant can acclimate to the given stress and acquire
tolerance or exhibit a hypersensitive response ultimately leading to programmed
cell death or necrosis. Consideration of the effect of stress beyond the individual
plant is also needed as such effects can lead to important short- or long-term shifts
in population and species persistence, potentially affecting biological diversity,
ecosystem functioning and carbon sequestration (Vickers et al. 2009a; Holopainen
et al. 2013).
Considerable research on the response of plants to stress has focussed on the
generic production of reactive oxygen species (ROS) such as singlet oxygen (1 O2 ),
superoxide (O2  ), hydrogen peroxide (H2 O2 ) and hydroxyl radicals (OH), because
they are important signalling molecules and serve to initiate defence responses (Apel
and Hirt 2004). Similarly, reactive nitrogen species (RNS), especially NO, which
are also generically produced under stress, are important signalling molecules,
especially activating hypersensitive responses to stress (Lamattina et al. 2003).
212 M. Possell and F. Loreto

Fig. 8.1 Plants are subjected to a variety of abiotic stresses. Often these stresses coincide with
each other, e.g., heat, high light and temperature or high temperatures and drought. The role of
constitutive BVOC biosynthesis and emission in maintaining or enhancing plant fitness during
stress events by increasing plant tolerance is still a matter of intensive investigations

Although constitutive isoprenoid emissions were initially considered to serve

in plant heat stress tolerance only (Singsaas et al. 1997), a general understanding
of the role of constitutive BVOC biosynthesis and emission in maintaining plant
fitness during stress events by increasing broad-spectrum tolerance to the production
of oxidative compounds has started to emerge (e.g., Vickers et al. 2009a; Loreto
and Schnitzler 2010). Much attention has been given to heat stress and drought,
reflecting the circumstance that both occur alone or in combination in many natural
ecosystems (Peñuelas and Llusià 2003). In this chapter, we will focus on these key
stress factors. We review the hypotheses of why plants emit certain constitutive
BVOCs with an emphasis on their capacity to enhance stress tolerance. This chapter
demonstrates the rich spectrum of BVOC emission responses to a variety of stresses
and argues that constitutive and induced emissions play key roles in providing plants
with broad tolerance to a diversity of stresses.

8.2 Abiotic Stresses: Effect on BVOC Biosynthesis

and Emissions

8.2.1 Temperature

Once produced, BVOCs partition between the gas and liquid phases within plants
according to their Henry’s law constant, but the equilibrium between the two
phases is determined by temperature (see Niinemets et al. 2004; Harley 2013).
8 The Role of Volatile Organic Compounds in Plant Resistance to Abiotic. . . 213

Consequently, as temperatures increase, more BVOC is expected to enter the gas

phase and be emitted from the plant. However, the anatomy of the leaf, especially
the presence of specialized storage structures (ducts or glands), and the existence of
diffusion resistances, can all modify BVOC emission. Although specialized storage
structures can contain large amounts of volatile terpenoids, the diffusion resistances
of cell walls of the storage structures are often large, such that plants with extensive
storage pools can be only moderate emitters (Kesselmeier and Staudt 1999;
Ghirardo et al. 2010), unless the pools are ruptured. Plants that do not store BVOCs
have small, temporary pools, and BVOCs diffuse out of the leaf according to their
concentration gradients. The limitation of the diffusion process by stomata depends
on compound physico-chemical characteristics. Emission of BVOC species with
a high Henry’s law constant such as isoprene and non-oxygenated monoterpenes
are weakly dependent on stomatal conductance (see Niinemets et al. 2004; Harley
2013). For these compounds, stomatal closure leads to a rapid increase in the partial
pressure of the BVOC, thereby almost instantly compensating for the increased
diffusion resistance. BVOCs that have a lower Henry’s law constant, such as
methanol or acetone, partition more into the liquid phase so that their emission rates
are affected for a longer time-span after reductions in stomatal conductance (see
Niinemets et al. 2004; Harley 2013). Changes in stomatal behaviour can occur for a
variety of reasons including heat stress or drought.
For de novo synthesized BVOCs in species lacking specialized storage structures,
temperature has a strong and immediate effect on emission rate. The response to
temperature is an Arrhenius type function with a maximum at temperature optimum,
beyond which catalysis and emission rates decline rapidly (see Grote et al. 2013;
Niinemets et al. 2010a for a review of these functions). However, there is often
a discrepancy between the optimum temperature at which the emissions are the
highest compared with the optimum temperatures that give the highest activity of
key rate-controlling enzymes such as isoprene synthase and monoterpene synthases.
This temperature optimum varies between 40 and 45 ı C for enzymes involved in the
biosynthesis of chloroplastic isoprenoids and is often a few degrees higher than
the emission optimum. This suggests that the temperature response of emission
is the combined function of both enzyme activity and substrate supply (Rasulov
et al. 2010).
With regard to isoprenoid compounds like isoprene, when temperatures increase
beyond the optimum required for peak emission, the production of photosynthetic
metabolites and energy needed for their synthesis is inhibited. This leads to a decline
in metabolite pool size and hence emission (Singsaas and Sharkey 2000; Magel
et al. 2006). If temperatures increase even further, heat-induced enzyme damage
can occur which means that the emission is not re-established to the original level
when temperatures are reduced (Loreto et al. 2006). However, the shape of the
temperature response curve can be modified by acclimation to different environ-
mental conditions. Acclimation of isoprene to high temperature may (Mayrhofer
et al. 2005; Wiberley et al. 2005; Vickers et al. 2010) or may not (Centritto et al.
2011) occur. This is currently a controversial issue with important ramifications for
accurate prediction of isoprenoid emission responses to temperature.
214 M. Possell and F. Loreto

Leaf temperatures have been observed to fluctuate within seconds and occasion-
ally exceed ambient temperatures by more than 10 ı C (Sharkey et al. 1996; Singsaas
et al. 1999). For enzymatically produced compounds like isoprene, the emissions do
not respond at the same frequency. In a study by Singsaas and Sharkey (1998),
the response of isoprene emissions to temperature was measured and two time
constants were identified. The first time constant of 8.2 s may reflect the influence of
temperature on isoprene synthase reaction kinetics, but it is too slow to fully respond
to the very rapid temperature fluctuations. The second time constant of 166 s is likely
related to regulation of substrate pool sizes and ultimately depends on changes in
the availability of energy, reducing power and carbon.
In addition to short-term extreme temperatures, sustained moderate heat stress
from tens of minutes to hours often leads to a reduction in isoprene emissions (e.g.,
Singsaas and Sharkey 2000) which can be related to reductions in metabolic activity
(e.g., Zhang et al. 2009) affecting the production of intermediate compounds for
isoprene biosynthesis (Niinemets et al. 2010b). As plants recover from prolonged
heat stress, isoprene emission capacities often increase, indicating acclimation to
past temperatures (Petron et al. 2001; Sharkey et al. 1999; Fig. 8.2a). Similar
responses have been observed in monoterpene emitters (e.g., Loreto et al. 1998;
Staudt and Bertin 1998), but the response can be moderated by the combined effect
of monoterpene synthase activity and the evaporation of non-specifically stored
monoterpenes (Ghirardo et al. 2010). In the case of monoterpenes that can be
stored in leaf lipid and liquid phases, non-specific storage may alter the temperature
responses (Niinemets and Reichstein 2003; Niinemets et al. 2010b).
Recent studies demonstrate that when the emission of volatile isoprenoids is
inhibited by heat stress, the emission of other BVOCs, in particular methanol and
GLVs, is enhanced (Loreto et al. 2006; Copolovici et al. 2012; Fig. 8.2b). The
emission of these compounds indicates damage of cell walls and membranes, and
elicitation of stress signalling pathways. Interestingly, the emission of GLVs was
observed to be sustained for the entire heating period and the emissions were
maintained long after temperature was returned to optimal levels. Like isoprenoids,
long-term changes in temperature can also affect the emission of GLVs. Work on
aspen (Populus tremula) where nighttime temperatures were elevated by 6–22 ı C in
4 ı C steps over a 6-week period, showed that GLV emissions increased significantly
during the day, peaking when night- and daytime temperatures were equal (Ibrahim
et al. 2010). In addition, an increase in the emissions of monoterpenes, sesquiter-
penes, and the homoterpene DMNT (4,8-dimethylnona-1,3,7-triene) emission were
observed (Ibrahim et al. 2010). In the same study, Ibrahim et al. (2010) discovered
that the emissions of DMNT, sesquiterpenes and GLVs from silver birch (Betula
pendula) were significantly increased by temperature. GLVs have not been the
only compound class noticed to have significant fluxes during heat stress. For
example, Schade and Goldstein (2001) measured enhanced methylbutenol, ethanol
and acetaldehyde fluxes from a ponderosa pine (Pinus ponderosa) plantation during
high temperature events.
8 The Role of Volatile Organic Compounds in Plant Resistance to Abiotic. . . 215

Fig. 8.2 Heat stress dependent changes in (a) isoprene and monoterpene constitutive emissions
and in (b) emissions of (E)-2-hexenal and methanol. Heat stress causes the emissions of isoprene
and monoterpenes to rise initially, but as the stress continues, the emissions decline. Upon stress
relief, the levels of constitutive isoprene and monoterpene emissions are often higher than before
the application of stress, indicating acclimation to stress. The responses of emissions of green leaf
volatiles and methanol to heat stress are different from isoprene and monoterpene emissions by
being sustained over the stress duration. The generalized responses in (a) and (b) are based upon
Loreto et al. (1998, 2006), Staudt and Bertin (1998), Velikova and Loreto (2005), and Magel et al.
(2006)

8.2.2 Drought

Drought is a key stress factor worldwide. During drought, stomatal conductance

is directly affected, and combined with enhanced mesophyll diffusion resistance,
causes substrate limitations to photosynthesis (Flexas et al. 2004). Intuitively, the
reduction of photosynthesis and stomatal conductance would be expected to nega-
tively impact BVOC emission by reducing the supply of carbon and energy to their
biosynthesis and/or by increasing the diffusional resistance to their emission. With
regard to isoprenoid emissions, drought effects primarily depend on the severity of
the drought (Fig. 8.3). Mild drought has been demonstrated to neither affect isoprene
(e.g., Pegoraro et al. 2004; Sharkey and Loreto 1993) nor monoterpene (e.g., Staudt
et al. 2002; Peñuelas et al. 2009) emissions. Once drought becomes prolonged or
heavy, isoprene and monoterpene emissions decline (e.g., Brilli et al. 2007; Peñuelas
et al. 2009; Sharkey and Loreto 1993; Staudt et al. 2002).
216 M. Possell and F. Loreto

Fig. 8.3 Drought stress dependent changes in constitutive isoprene or monoterpene emissions.
Mild drought stress does not affect emissions, but as the stress continues, reductions in emission
occur. Upon re-watering, the level of emission is often higher than before the imposition of stress,
indicating an acclimation to the stress. The generalized response is based upon Brilli et al. (2007),
Peñuelas et al. (2009) and Bertin and Staudt (1996)

8.2.2.1 Isoprene Emissions Under Drought

How can isoprene emissions be sustained under drought stress? Schnitzler et al.
(2004) and Loreto et al. (2004a), using 13 C-labelling techniques, have demonstrated
that while the photosynthetic carbon input is curtailed, alternative carbon sources
from starch breakdown or respiration can compensate for reductions in the primary
carbon source. Indeed, work by Brilli et al. (2007) has shown that there is a
preference for ‘old’ unlabelled (12 C) carbon over recently fixed, 13 C-enriched
photosynthetic intermediates when photosynthesis and isoprene production become
uncoupled during drought stress. The use of this alternative carbon may also explain
enhanced isoprene emission rates after re-watering that have been observed in some
studies (Sharkey and Loreto 1993; Peñuelas et al. 2009). Once isoprene emission
and photosynthesis are re-coupled, the use of alternative carbon sources rapidly
ceases (Brilli et al. 2007).
The need to consider the CO2 response of isoprene (Grote et al. 2013; Monson
2013) is particularly important under plant stress. Niinemets et al. (2010b) have
used the shape of the isoprene-CO2 response as a way to explain isoprene emissions
under different drought conditions. In this paper, they argue that the severity of
a particular drought event can result in different Ci values and hence different
isoprene emission rates. When mild drought conditions occur, they state that Ci
will decrease by approximately 50–80 mol mol1 . A reduction of this magnitude
has a small positive effect on isoprene emissions and depending upon the species,
may result in no observable changes in emissions or a slight increase. When severe
drought occurs, Ci values drop to close to the photosynthetic compensation point,
which is where isoprene emissions drop off dramatically. This view may be an
oversimplification because (a) when already photosynthetic rate-limiting proteins
are downregulated, there might be a reduced sink for CO2 and CO2 can accumulate
in the intercellular spaces due to respiration. However, this is common only when the
8 The Role of Volatile Organic Compounds in Plant Resistance to Abiotic. . . 217

stress is severe and isoprene is probably already limited by substrate availability; (b)
low CO2 acquisition through stomata may be in part compensated by refixation of
CO2 produced by other sources, especially mitochondrial respiration. Standard gas-
exchange methodologies allow calculation of the Ci without discerning its carbon
sources, but refixed CO2 does likely contribute to isoprene emission (Loreto et al.
2004a). Furthermore, changes may occur in isoprene synthase activity and source
of carbon supply under extreme drought (Brilli et al. 2007; Fortunati et al. 2008).
Overall, the existence of a negative correlation between Ci and isoprene emission is
now unambiguous, even in un-stressed plants (Guidolotti et al. 2011).
A question remains as to whether the maintenance of isoprene production under
drought stress improves plant function and fitness? Studies in transgenic Arabidop-
sis plants containing an isoprene synthase gene concluded that the presence of the
trait did not increase drought resistance (Sasaki et al. 2007). Work by Fortunati
et al. (2008) showed that during severe drought stress and during the recovery
from stress, the temperature dependency of isoprene emission is modified. Although
not explicitly demonstrated, Fortunati et al. (2008) postulated that alteration of the
temperature response likely resulted from changes in substrate availability and/or
isoprene synthase protein concentrations.

8.2.2.2 Drought Effects on Monoterpenes and Oxygenated

Volatile Emissions

The effect of drought stress on monoterpene emissions has not been as intensively
studied as drought effects on isoprene emissions. Nevertheless, there are field and
laboratory observations showing that inhibition of monoterpene emission occurs
under severe drought stress, while the emission rates are maintained under less
severe stress (e.g., Peñuelas et al. 2009; Bertin and Staudt 1996; Šimpraga et al.
2011). As with isoprene, differential availability of carbon substrates may explain
these observations. However, drought may change qualitatively the composition
of the monoterpene blend because of differences in the Henry’s law constant of
monoterpenes. In practice, non-oxygenated terpenes are unaffected by moderate
drought, while the emissions of oxygenated volatiles are strongly curbed (Niinemets
et al. 2002; Harley 2013).
In terpene-storing species, the effect of drought on monoterpenes might depend
upon the season. In a study on Mediterranean woody species, Llusià et al. (2006)
showed that leaf concentrations of monoterpenes were greatest in winter and lowest
in summer, but responses during drought were highly variable. For example, in
Aleppo pine (Pinus halepensis), the concentrations decreased in response to drought
in winter and increased in summer. In contrast, drought decreased monoterpene
concentrations in summer and increased them in winter in Pistacia lentiscus. The
differences in concentrations were associated with different drought responses of
emissions. Emission rates of monoterpenes from Mediterranean shrublands were
more strongly affected by drought during spring and summer than during autumn
218 M. Possell and F. Loreto

and winter (Llusià et al. 2008). Even fewer studies have been done on drought
effects on sesquiterpenes, but these studies indicate a rapid decline in emissions
with increasing drought (Ormeño et al. 2007).
Oxygenated BVOCs, such as methanol or acetone, partition more into the liquid
phase than the gas phase (Niinemets et al. 2004). Consequently, stomatal closure
in response to drought and salt stress would be expected to reduce the emission of
these compounds and affect the gaseous emissions over a much longer time-span
than the emissions of isoprene or non-oxygenated volatiles (see Niinemets et al.
2004; Harley 2013). However, even in plants subject to drought stress, morning
peaks of methanol and acetone emission, coincident with stomatal opening, have
been observed (Fares et al. 2009; Filella et al. 2009) corresponding to “outgassing”
of a large liquid-phase pool built up during the night when stomata were closed
(Niinemets and Reichstein 2003; Harley et al. 2007; Harley 2013). Green leaf
volatiles can be formed also under severe drought if the stress leads to the cell wall
and membrane damage (Capitani et al. 2009), although there are much less data for
drought effects on GLV than for the effects of other stresses.

8.2.3 Salinity Stress

The effect of salinity on photosynthesis and stomatal conductance is similar to

that of drought with significant decreases observed as salinity increases (Loreto
and Delfine 2000; Teuber et al. 2008). Therefore, like drought, the reduction of
photosynthesis and stomatal conductance would be expected to negatively impact
BVOC emission by either reducing the supply of carbon and energy to their
biosynthesis or by increasing the diffusional resistance to their emission. However,
isoprene emission rates measured under conditions of transient or continual salinity
stress were found to be unaffected relative to controls (Loreto and Delfine 2000;
Teuber et al. 2008). In contrast to the isoprene emission rates, Teuber et al. (2008)
showed that in grey poplar (Populus x canescens), continuous salinity stress led to a
decrease of the leaf concentrations of the immediate substrate for isoprene synthesis,
dimethylallyl diphosphate (DMADP). They argued that this decline is probably
due to the limited availability of photosynthates for isoprene biosynthesis (Teuber
et al. 2008). Thus, the high demand of isoprene synthase for its substrate in salt-
stressed plants results in an increased turnover rate of DMADP in the salt-stressed
plants. Analogous to isoprene, acetaldehyde, formaldehyde and acetone emission
rates were not influenced by salinity in this study (Teuber et al. 2008).
Very few studies have looked at the effect of salinity on monoterpenes. In
perennial evergreen sage (Salvia officinalis), leaf monoterpene contents were found
to increase with increasing salinity, but once the salinity reached a threshold level,
monoterpene content began to decline (Ben Taarit et al. 2009). The same study
also found that the composition of the monoterpenes changed depending upon the
level of salinity. Similar results have been found in marjoram (Origanum majorana)
(Jelali et al. 2011).
8 The Role of Volatile Organic Compounds in Plant Resistance to Abiotic. . . 219

8.2.4 Combined Stresses

In addition to heat, drought and salinity, plants can be subjected to a number of other
environmental stresses. The effects of flooding on BVOC emissions are reported in
the chapter of Kreuzwieser and Rennenberg (2013), and the effects of air pollution in
chapters of Calfapietra et al. (2013) and Holopainen et al. (2013). However, stresses
often occur in combination, for example, drought may occur with increased salinity
and/or heat or biotic stress can also occur with abiotic stress (Loreto and Schnitzler
2010; Niinemets 2010a). Although we could predict this behaviour based upon the
known responses for each factor, the response by a plant can be unique and not
directly extrapolated from the response of plants to each of the stresses applied
individually (Mittler 2006; Niinemets 2010b). For instance, important modifications
in the temperature response of isoprene emission rates under drought have been
observed. Individually, an increase in drought and temperature would normally lead
to an increase in isoprene emission, but Fortunati et al. (2008) discovered that,
after drought, the plants in higher temperature treatment had suppressed emissions
for at least for 2 weeks (i.e., leaves from 25 and 35 ı C treatments showed the
same isoprene emission rates), despite photosynthesis quickly returned to pre-stress
levels. Work by Centritto et al. (2011) not only supported this finding but also
demonstrated that increases in temperature cannot offset the inhibition of isoprene
under water-stress. This highlights the need for more multi-factorial studies focusing
on BVOC biosynthesis and emission and leads us to question how the biosynthesis
and emission of constitutive BVOCs can enhance stress tolerance, especially in
multi-stress situations?

8.3 How Do BVOCs Enhance Stress Tolerance?

Obviously, the constitutive biosynthesis and emission of BVOCs must benefit the
plant in some way. As highlighted above, the biosynthesis and emission of BVOCs,
especially isoprene, appears to be tuned to changes in the level of abiotic stress that
a plant is exposed to. If the plant is benefiting from volatile emissions in some way,
how is it benefiting and through what mechanism (Fineschi et al. 2013)? Studies of
transgenics either engineered to emit isoprene (Sasaki et al. 2007; Velikova et al.
2011; Vickers et al. 2011) or to knock out isoprene (Behnke et al. 2007; Rosenkranz
and Schnitzler 2013) have conclusively indicated improved abiotic stress tolerance
in isoprene emitting genotypes. These studies are reviewed in detail in the chapter
of Rosenkranz and Schnitzler (2013). Here we analyse the evidence from both wild,
and to some extent transgenic plants that highlights the specific mechanisms via
which isoprene enhances abiotic stress resistance.
As a consequence of the dominance of isoprenoids, and in particular isoprene, in
BVOC emissions (e.g., Guenther et al. 1995) the majority of studies of different
stress factors have also concentrated on isoprene. This was best illustrated by
Peñuelas and Staudt (2010) were they showed that research into isoprene dominated
220 M. Possell and F. Loreto

all the potential environmental stress factors with respect to global and climate
change drivers. Given the wealth of the volatiles released by plants, we echo the
plea for more detailed studies of other key BVOCs.

8.3.1 Membranes

One of the oldest hypotheses as to why plants produce isoprene is that it enhances
the thermotolerance of the photosynthetic apparatus by stabilising the chloroplastic
(thylakoid) membranes under transient heat shocks (Sharkey and Singsaas 1995).
This was based upon two main observations. First, higher rates of photosynthesis
and electron transport were measured from plants fumigated with isoprene when
they were exposed to rapidly increasing temperatures. Second, previous reports
indicated that other isoprenoid compounds are important in membrane physiology
(Ourisson and Nakatani 1994). Subsequent experiments have confirmed that iso-
prene does confer thermotolerance to photosynthesis (e.g., Sharkey et al. 2001;
Velikova et al. 2006; Behnke et al. 2007, 2010; Way et al. 2011) and this has also
been demonstrated with light-dependent monoterpene emissions (Loreto et al. 1998;
Delfine et al. 2000) but only for certain monoterpenes (Copolovici et al. 2005). In all
of these studies, photosynthetic rates measured after stress were significantly higher
than in plants with suppressed isoprene or monoterpene emission rates.
In silico studies by Siwko et al. (2007) indicated that isoprene partitions into
the centre of the phospholipid membranes and maintains their order without
significantly changing the dynamic properties of the membrane (Fig. 8.4a). Isoprene
occupies the volume between the lipid tails thereby increasing the adhesive forces
and acting as molecular glue. As temperatures decrease, an exponential decline
in isoprene biosynthesis would concurrently occur preventing solidification of the
membrane. Conversely, an increase in temperature would lead to more isoprene
and greater membrane stability. Consequently, Siwko et al. (2007) concluded that
isoprene-producing plants could maintain membrane in functional liquid-crystalline
phase and photosynthetic activity over a wider temperature range. However, Logan
et al. (1999) failed to demonstrate isoprene protection on fluidity or functions
of reconstituted membranes. Indirect evidence that isoprene stabilises thylakoid
membranes comes from the use of inhibitors of isoprene production even in the
absence of heat stress. However, Possell et al. (2010) showed that the application of
fosmidomycin to inhibit isoprenoid production led to significant reduction in the
operating efficiency of photosystem II. Recently, laboratory studies by Velikova
et al. (2011), using three different techniques, showed that isoprene improves the
integrity of the photochemistry of photosynthesis under heat-stress conditions by
affecting large-scale thylakoid membrane organisation. The authors also demon-
strated that a continuous biosynthesis of isoprene was necessary for this to occur,
which would be consistent with the need for a volatile substance that could rapidly
escape after transient heat shocks to prevent transition of membranes from liquid-
crystalline phase to solid-gel phase (Siwko et al. 2007).
8 The Role of Volatile Organic Compounds in Plant Resistance to Abiotic. . . 221

Fig. 8.4 Schematic overview of the proposed metabolic functions of volatile terpenes: (a)
stabilisation of phospholipid membranes under heat stress by isoprene as indicated by in silico
modelling (Siwko et al. 2007); (b) ‘metabolic overflow hypothesis’ as proposed by Rosenstiel et
al. (2004) and (c) the ‘single biochemical mechanism for multiple physiological stressors’ model
proposed by Vickers et al. (2009a). The model shows how volatile isoprenoids may exert protective
effects through antioxidant activity. In (b), the negative sign indicates the impact of an increase
(positive sign) in phosphoenol pyruvate carboxylase (PEPc) activity on isoprenoid biosynthesis
and emission. DMADP – dimethylallyl diphosphate, GAP – glyceraldehyde 3-phosphate, MTs –
monoterpenes, OAA – oxaloacetate, PEP – phosphoenol pyruvate, PEPc – phosphoenol pyruvate
carboxylase, PK – pyruvate kinase, Pyr – pyruvate, TP – triose phosphate. In (c), PCD –
programmed cell death, RES – reactive electrophilic species, RNS – reactive nucleophilic species,
ROS – reactive oxygen species

8.3.2 Role as Antioxidants

Under stress conditions, a variety of ROS are produced within plant cells, which
once left detoxified, can cause significant damage. Plants have a well-developed
antioxidant system consisting of liphophilic (e.g., tocopherols and carotenoids), hy-
drophilic (e.g., ascorbate and glutathione) and enzyme components which together
mediate the effect that ROS have upon the plant cell. The production of ROS even
under non-stressed conditions is an integral part of photosynthetic apparatus. Hence,
222 M. Possell and F. Loreto

ROS concentrations are tightly regulated by scavenging excess ROS to prevent

cytotoxic effects (Apel and Hirt 2004). Experiments with inhibitors of isoprene
biosynthesis and those using transgenic plants have demonstrated that the presence
of isoprene keeps ROS and the levels of lipid peroxidation to a level much lower
than isoprene-inhibited or non-emitting plants (e.g., Loreto and Velikova 2001;
Velikova et al. 2006, 2012; Vickers et al. 2009b). These findings indicate that volatile
isoprenoids have a role in moderating the oxidative load under stress conditions.
How volatile isoprenoids moderate oxidative load is still a matter of debate, but
the current evidence points to two main avenues: either direct reactions between
volatile isoprenoids and oxidizing species or mediation of the signalling responses
(Fig. 8.4c). Many BVOCs, especially isoprenoids, contain double bonds which
make them susceptible to oxidation via reactions with ROS. When an oxidizing
agent, such as ozone, enters a leaf, it reacts with reactive hydrocarbons, but it can
also elicit a cascade of reactions potentially resulting in formation of other damaging
ROS. Thus, a capacity to rapidly regulate plant oxidative status is essential. In this
regard, it is relevant that hydroxyl radicals react with isoprene in the aqueous phase
to form 2-methyltetrols (Santos et al. 2006). Isoprene is also known to protect
against singlet oxygen (Affek and Yakir 2002; Velikova et al. 2004) which is
produced at the thylakoid membranes when absorbed energy is in excess of that
used in photosynthesis (Asada 2006). This may occur because excess light is present
at high light intensities and/or because the use of excitation energy is retarded by
various abiotic stresses (Vickers et al. 2009a).
Mediation of signalling responses is the other possible mechanism by which
BVOCs such as isoprenoids can contribute to protective effects. Isoprene is oxidized
to methyl vinyl ketone (MVK) and methacrolein (MAC) in the atmosphere (Fuentes
et al. 2000), and the same compounds have been recently demonstrated to appear
when isoprene is oxidized within leaves (Jardine et al. 2011). Both MVK and
MAC are reactive electrophilic species (RES) and, along with unsaturated carbonyl
compounds such as GLVs like (E)-2-hexenal, are known to stimulate the expression
of defence genes (Almeras et al. 2003) (Fig. 8.2c). Similarly to isoprene, nitric oxide
(NO) is involved in a number of stress-related physiological processes including the
direct scavenging of ROS (Beligni and Lamattina 2002) and the plant hypersensitive
response (Gould et al. 2003; Wilson et al. 2008). Interestingly, isoprene-emitting
leaves produce less NO compared to isoprene-inhibited leaves when subjected to
oxidative stress (Velikova and Loreto 2005; Velikova et al. 2008). Although the exact
mechanism for these observations is unknown, any change in NO concentration
can potentially mediate the hypersensitive response and the onset of programmed
cellular death.
Unlike exposure to ozone or heat stress, there does not appear to be any evidence
that isoprene or monoterpenes protect against the consequences of flooding. ROS
formation in leaves with flooded root systems has been shown to play a role in
flooding-driven decline in plant physiological activity (Visser et al. 2003; Yordanova
et al. 2004; Kreuzwieser and Rennenberg 2013). Thus, isoprenoids could play
an important role in quenching ROS in flooded plants. However, Copolovici and
8 The Role of Volatile Organic Compounds in Plant Resistance to Abiotic. . . 223

Niinemets (2010) demonstrate that the capacity for isoprene emission was not linked
to flood tolerance in three species studied. Isoprene emission was lacking in the most
flood-tolerant species black alder (Alnus glutinosa), while the species with highest
isoprene emission rate red oak (Quercus rubra), was most intolerant of flooding and
could not sustain high isoprene emission rates under flooding.

8.3.3 Safety Valve

A number of studies have observed that the emission of isoprene is strongly

correlated with photosynthetic electron transport (e.g., Possell et al. 2004) and hence
leaf ATP content (Loreto and Sharkey 1993; Rasulov et al. 2009b). On a molar basis,
a considerable amount of photosynthetic energy is utilised in the biosynthesis of
isoprene (20 ATP, 14 NADPH; Sharkey and Yeh 2001). In reality, the consumption
of energy for isoprene production under non-stressed conditions is relatively small.
The emission of isoprene is measured in nmol m2 s1 , and the emissions are
typically an order of magnitude smaller than the rate of photosynthesis. Sharkey
et al. (2008) estimated that for an isoprene emission rate set at 2 % of photosynthesis
only 2.7 % of ATP and 3.4 % of NADPH is required for isoprene biosynthesis
compared to the 20–40 % used in photorespiration. Based upon these calculations
Sharkey et al. (2008) argued that the postulation by Magel et al. (2006) that the
function of isoprene emission was to dissipate unused energy does not hold up to
quantitative scrutiny. However, if under high temperature and high light, up to 50 %
of the isoprene produced in the chloroplast is actually oxidised to MVK and MCR
prior to leaving the leaf, then the sink for electrons would be much higher (Jardine
et al. 2011).
Rosenstiel et al. (2004) hypothesised that isoprene synthase, and its associated
isoprene emission, is a mechanism for balancing the demand for DMADP (by
higher isoprenoid biosynthesis) against the supply of DMADP (Fig. 8.4b). This
would ensure that chloroplastic DMADP will be available for the crucial synthesis
of carotenoids and chlorophyll when it is needed, and isoprene synthase prevents
DMADP from accumulating to levels that unnecessarily sequester phosphate.
According to this hypothesis, cytosolic phosphoenol pyruvate carboxylase plays a
pivotal role in dividing the phosphoenol pyruvate pool between isoprene biosyn-
thesis and other forms of carbon metabolism (Fig. 8.2b). Sharkey et al. (2008)
argued that because of the sensitivity of the first enzyme in the MEP pathway to
feedback from other metabolites of the MEP pathway, there is no need for this type
of regulatory mechanism. Although methods to investigate MEP pathway substrates
exist (e.g., Rasulov et al. 2009a) and information on how the MEP pathway is
regulated is being ascertained (e.g., Rivasseau et al. 2009; Mongelard et al. 2011; Li
and Sharkey 2013 a, b), it is still not entirely clear how feedback modulation of the
MEP pathway operates and by how much chloroplastic DMADP fluctuates.
224 M. Possell and F. Loreto

8.3.4 Indirect Consequences on Ecosystem Engineering

Hastings et al. (2007) defined ecosystem engineering as a concept that focuses on

how organisms physically change the abiotic environment and how this feeds back
to the biota. The concept applies well to the feedbacks between BVOCs produced by
the ecosystems and the atmosphere. A consequence of the emission of BVOCs into
the atmosphere is that their oxidation leads to the formation of secondary organic
aerosols (SOA). The formation of SOA has been demonstrated in both the laboratory
(O’Dowd et al. 2002; Joutsensaari et al. 2005; Hao et al. 2009; Kiendler-Scharr et al.
2009; Mentel et al. 2009; Virtanen et al. 2010) and forests for isoprene (Claeys et al.
2004), monoterpenes (Tunved et al. 2006; Kiendler-Scharr et al. 2009; Virtanen et al.
2010), sesquiterpenes (Boy et al. 2008), oxygenated VOCs (Mentel et al. 2009)
and GLVs (Hamilton et al. 2009). Aerosols modify the Earth’s radiative balance
though direct scattering of radiation, the formation of cloud condensation nuclei that
lead to enhanced cloud albedo, or even absorbing energy and promoting warming
(Ramanathan et al. 2007; Spracklen et al. 2008; Kulmala et al. 2013).
Vegetation is sensitive to changes in radiation, in particular to the diffuse
and direct fractions of sunlight. Roderick et al. (2001) showed that increases in
particulate matter increased the amount of diffuse radiation and this penetrated
inside tree canopies enhancing the overall photosynthetic rate. Therefore, any stress
that leads to changes in the emission of BVOCs has the potential to affect SOA
formation, radiative forcing and photosynthetic rates (Kulmala et al. 2013). As
woodland landscapes are estimated to contribute to 75 % of the total annual BVOC
emissions (Guenther et al. 1995) any rapid change in BVOC emission has the
massive potential to rapidly influence, or engineer, their surrounding environment.
However, the potential benefits of SOA formation from BVOCs for the ecosystem
have to be balanced against the potential negative effects of BVOC oxidation, such
as ozone formation, and the consequences these effects have upon the ecosystems
(Lerdau 2007).

8.3.5 Multifunctional Compounds?

Vickers et al. (2009a) argued that volatile isoprenoids played an important role in the
protection against abiotic stresses by helping plants improve their ability to deal with
oxidative changes regardless of the nature of the stressor. The authors suggest that
the activity of ROS is mediated through the combined action of direct reactions with
ROS species and the indirect consequences this has for ROS signalling. The authors
also state that membrane stabilisation decreases lipid peroxidation, thus directly
impacting the oxidative status of the cell. Indeed, Velikova et al. (2012) argue that
the stabilisation of the thylakoid membranes by isoprenoids may be the reason
for reduced ROS levels observed in heat-stressed plants. Until these questions are
resolved, there will be a continued debate within the BVOC community as to the
primary function of isoprene. However, the biosynthesis of isoprene may actually
8 The Role of Volatile Organic Compounds in Plant Resistance to Abiotic. . . 225

be multifunctional. A compound that can stabilise membranes against transient heat

stress and minimise ROS production, remove ROS through direct reactions and
through its reaction products, generate reactive electrophilic species (RES) that
initiate the expression of defence genes, while at the same time modifying the
atmosphere to enhance photosynthetic light use efficiency, is certainly worth the
substantial investment in carbon and energy.

8.4 Evolution of the Stress Tolerance Function

Plants produce a wide range of BVOCs but not all plants produce the same BVOCs
and it remains to be seen if plants have the capacity to produce a wider range of
BVOCs than those measured so far. In the sections above, we saw evidence that
isoprene biosynthesis and emission play a significant role in tolerating heat, drought
and oxidative stress induced by pollutants. A puzzling issue of isoprene in stress
tolerance is that given the many benefits isoprene confers for stress tolerance, why
not all plants have evolved the capacity to produce it? Isoprene emitters can be found
among mosses (Hanson et al. 1999), ferns (Tingey et al. 1987) and in gymnosperms
and angiosperms (see http://bai.acd.ucar.edu/Data/BVOC/for a comprehensive list
and (Fineschi et al. 2013) for a detailed discussion), but not all species of a particular
genus make isoprene. For example, species in the sub-genus Sclerophyllodrys from
the genus Quercus are light-dependent monoterpene emitters, while the remaining
sub-genera (except for species from sub-genus Cerris, which are predominantly
non-emitters) are isoprene emitters (Loreto 2002).
Evolutionary aspects of BVOC emission have been discussed in some detail in
several other chapters in this book (Fineschi et al. 2013; Li and Sharkey 2013; Rajabi
Memari et al. 2013). Monson et al. (2013) hypothesise that there is a narrow range of
conditions in which isoprene is advantageous as protection against stress. Dealing
with the stress tolerance function of isoprene, we only surmise here that changes
in atmospheric CO2 concentrations might have been a selective filter, modifying
selection pressures for or against the evolution of isoprene biosynthesis. Recent
studies by Darbah et al. (2010) and Way et al. (2011) into thermotolerance under
different [CO2 ] regimes suggested greater tolerance under low [CO2 ] conditions
than elevated [CO2 ] conditions. Thus, Way et al. (2011) argued that the evolutionary
pressure that led to the independent evolution of isoprene emission was a low [CO2 ]
atmosphere. This supports the views of Sharkey and Yeh (2001) that the evolution
of isoprene emission was to cope with environmental conditions not conducive
to photosynthesis. Indeed, there has been a number of epochs in Earth’s history
where CO2 concentrations have been lower than the current concentration and such
conditions can make photosynthesis more susceptible to heat and light damage
(Cowling and Sage 1998).
We must also consider that selective pressures could lead to the loss of
isoprene emission in different genera and epochs. For example, if a low CO2
concentration could lead to the evolution of isoprene biosynthesis, then an elevated
226 M. Possell and F. Loreto

[CO2 ] atmosphere could lead to its loss. Evidence from long-term [CO2 ]-isoprene
studies does show a suppression of isoprene emissions by reducing both substrate
availability and enzyme activity (Scholefield et al. 2004; Possell and Hewitt
2011; Calfapietra et al. 2013) (but see Sun et al. 2012). However, explanations
of the patterns of why plant lineages do or do not emit isoprene also have to
consider complex interactions with other environmental factors, plant phenotypes
and patterns of geographic migration (Monson et al. 2013).
The mass production of a single, volatile compound, particularly over a long
period of time would be an inefficient way to sustain protection. Indeed, there
is no evidence to date that isoprenoid emitting species cope better in the long-
term in the presence of environmental stresses. However, the production of reactive
electrophilic species (RES) from the oxidation of volatile isoprenoids could lead to
the biosynthesis of efficient mechanisms for protection or defence (e.g., Almeras
et al. 2003) which can result in acclimation and tolerance to a particular stress. For
example, other known thermotolerance mechanisms, such as changes in xanthophyll
epoxidation state, synthesis of heat shock proteins, and changes in membrane lipid
composition, can take many minutes, hours, or days to respond to a temperature
episode (Singsaas and Sharkey 1998). Sustained stress results in the reduction of
constitutive BVOCs in most cases (see above) and only the prolonged fumigation
with ozone can lead to a sustained increase in emissions (Velikova et al. 2005a).
Once the stress has been relieved, however, increases in emissions have been
observed. This has been observed after heat stress (Velikova et al. 2005b), drought
(Sharkey and Loreto 1993; Pegoraro et al. 2004; Peñuelas et al. 2009), salinity
(Loreto and Delfine 2000; Teuber et al. 2008), and ozone (Loreto et al. 2004b).
Niinemets (2010a) suggested that the previous condition now constitutes a new mild
stress for the leaves acclimated to the stressed environment, explaining maintenance
of the higher emissions, or it may prime the defensive apparatus to maintain an
active antioxidant system in order to rapidly react to other forthcoming stresses.

8.5 Summary and Conclusions

A large number of stress factors affect the emission of BVOCs and the existing
tolerance, timing, duration and severity of the stresses is known to mediate the
magnitude of the emissions of constitutive BVOCs or induce de novo synthesis of
new induced BVOCs (e.g., Loreto et al. 2006). In this chapter we have seen that
this is especially true for the biosynthesis and emission of isoprenoids, oxygenated
BVOCs and GLVs in response to heat and drought stress. Research in the role of
BVOCs in stress tolerance has predominantly concentrated on isoprene, and to a
lesser extent monoterpenes, but substantially more research is required with regard
to other BVOCs that may be more difficult to detect and to better understand the role
other isoprenoids and non-isoprenoid compounds undertake in stress tolerance and
acclimation. The development of transgenic plants that have had isoprene synthase
genes added or silenced is helping us understand the role that isoprene plays in
8 The Role of Volatile Organic Compounds in Plant Resistance to Abiotic. . . 227

stress tolerance and the mechanism by which it performs this function (Rosenkranz
and Schnitzler 2013). Similar developments in identifying genes responsible for
other BVOCs would be a necessary next step in assessing their potential in plants
acquiring stress tolerance to abiotic stress, biotic and multi-factorial stresses, and in
maintaining fitness and survival. Such research may allow us to identify common
mechanisms, such as the one suggested by Vickers et al. (2009a) for isoprenoids,
which will enable us to improve and simplify our understanding of plant responses
to stress. These developments could aid in the improvement of models of BVOC
emissions under transient and complex environmental conditions, allowing for
better assessment of the impact BVOCs have upon atmospheric chemistry, climate
and their associated feedbacks.

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Chapter 9
Flooding-Driven Emissions from Trees

Jürgen Kreuzwieser and Heinz Rennenberg

Abstract Anoxia in the root system leads to the formation of ethanol in roots,
the transport of ethanol to the leaves and strong foliar emissions of ethanol and
acetaldehyde. In addition, emissions of typical stress-related volatiles are elicited.
This chapter reviews the environmental, biochemical and physiological controls on
flooding-driven products of anoxic metabolism and stress signalling compounds.
It demonstrates that the various controls operate at different timescales and,
furthermore, that these emissions are characterized by strong differences between
species.

9.1 Introduction

Higher plants are aerobic organisms depending on a continuous supply of oxygen

which is required to maintain crucial physiological processes such as mitochondrial
respiration (Drew 1997). Nevertheless, if adverse environmental stress factors lead
to diminished or even completely reduced oxygen availability, plants can survive
for a limited period of time. The fate of plants stressed by anoxia (complete lack of
oxygen) or hypoxia (a limited amount of oxygen is still available) strongly depends
on plant species and age, season, duration, and severity of the stress event, and other
environmental conditions during stress (Drew 1997). Depending on these factors,
plants can survive stress periods for up to several months or are severely injured after
only some hours under stress (Drew 1997; Vartapetian and Jackson 1997). Stress
tolerance can be a consequence of (i) morphological adaptations helping to avoid
or delay the occurrence of oxygen deprivation and/or (ii) changes at the metabolic
level required to endure the period of reduced oxygen supply (Bailey-Serres and

J. Kreuzwieser () • H. Rennenberg

Professur für Baumphysiologie, Albert-Ludwigs-Universität Freiburg, Georges-Köhler-Allee
Geb. 053/054, D-79110 Freiburg im Breisgau, Germany
e-mail: [email protected]; [email protected]

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 237
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 9,
© Springer ScienceCBusiness Media Dordrecht 2013
238 J. Kreuzwieser and H. Rennenberg

Voesenek 2008). Several omics approaches with anoxia-tolerant and -sensitive

herbaceous and tree species have indicated that metabolic adaptations to anoxia
or hypoxia need well-orchestrated changes in numerous physiological processes,
including the onset of fermentative processes, a stimulated flux through glycolysis,
and the down-regulation of energy demanding processes (Klok et al. 2002; Liu et al.
2005; Loreti et al. 2005; Lasanthi-Kudahettige et al. 2007; Kreuzwieser et al. 2009;
Licausi et al. 2010).
Because the lack of oxygen dramatically affects mitochondrial respiration,
the plants’ main process of ATP production, energy metabolism is strongly
impaired by anoxia or hypoxia. The ability to induce alternative ATP generating
pathways in stress-exposed tissues is therefore essential for the plants’ survival. In
particular, fermentative pathways play a crucial role in the acclimation to flooding
(Crawford and Finegan 1989; Drew 1997; Bailey-Serres and Voesenek 2008). As
a first response to reduced oxygen availability, several plant species induce lactic
acid fermentation. Because accumulation of lactic acid considerably reduces the
cytoplasmic pH, this pathway can only be maintained transiently and rapidly has
to be replaced by alcoholic fermentation (Davies 1980; Roberts et al. 1984). The
energy (ATP) yield of fermentative pathways is only 2 mol ATP per mol glucose
consumed. This is very inefficient compared to the energy yield of mitochondrial
respiration of up to 38 mol ATP per mol glucose consumed. Many changes observed
at the metabolome and transcriptome level induced by flooding can be explained by
impaired energy metabolism and the need to maintain energy supply (Bailey-Serres
and Voesenek 2008). Consequently, several energy-consuming processes such as
transport of nutrients, biosyntheses of cellulose, lignin and other macromolecules,
and growth are strongly inhibited under anoxia (Loreti et al. 2005; Lasanthi-
Kudahettige et al. 2007; Kreuzwieser et al. 2009). Surprisingly, clear differences
between anoxia-tolerant and -sensitive plant species at the metabolic level have
not been identified so far (Kreuzwieser et al. 2009). Independent of the species’
flood tolerance, energy-consuming pathways are slowed down, while the glycolytic
flux – leading to ATP production during fermentation – is enhanced (Klok et al.
2002; Liu et al. 2005; Loreti et al. 2005; Branco-Price et al. 2005; van Dongen et
al. 2008; Kreuzwieser et al. 2009). Major differences between flood-tolerant and
-sensitive species include a much higher number of differentially expressed genes
in flood-tolerant compared to -sensitive species and modified temporal patterns
of gene expression (Kreuzwieser et al. 2009). For example, in poplar, a flood-
tolerant species, a maximum of 5,000 genes with altered expression was observed
after 14 days of flooding (Kreuzwieser et al. 2009) whereas in flood-sensitive
Arabidopsis, a maximum of only ca. 1,500 genes with altered expression was found
after 2–4 h of anoxia; afterwards the number of differentially expressed genes even
declined in Arabidopsis (Liu et al. 2005).
The present chapter summarizes recently gained knowledge of flooding-induced
volatile emissions. It examines the physiological determinants of flood tolerance
with main emphasis on flooding-induced volatile metabolic intermediates and stress
marker compounds. Although flooding stress is a key limiting factor in a number
of ecosystems, the reasons for interspecific variations in flood tolerance and also
9 Flooding-Driven Emissions from Trees 239

in flooding-driven volatile release are still poorly understood. Here we argue that a
plant internal cycling of ethanol might contribute to flood tolerance of trees. Ethanol
is produced in anoxic roots of trees. It is then transported with the transpiration
stream to the leaves and oxidized in the leaves to acetaldehyde and acetic acid as
volatile intermediates. The main portion of the ethanol is, however, used in the
leaves’ primary carbon metabolism thereby allowing the use of the energy-rich
reduced carbon of this fermentation product.

9.2 Products and Intermediates of Alcoholic Fermentation

9.2.1 Key Fermentation Products Emitted from Flooded Plants

The emission of volatile compounds from leaves of flooded plants is closely

connected to the altered metabolism in anoxia exposed roots. Several studies have
demonstrated that the end-product of alcoholic fermentation, ethanol, is loaded into
the xylem and transported with the transpiration stream to the leaves of the plants
(Crawford and Finegan 1989; MacDonald and Kimmerer 1991; Kreuzwieser et al.
1999). In the leaves, only a small part, a few percent of the ethanol delivered is
emitted into the atmosphere. The main part, however, is oxidized in the leaves
(MacDonald and Kimmerer 1993; Ferner et al. 2012), and the oxidation products are
also either emitted or metabolized by the plant. In particular, the first intermediate
of ethanol oxidation, the relatively volatile compound acetaldehyde, is also partially
emitted into the atmosphere (Kreuzwieser et al. 1999; Ferner et al. 2012). The
majority of acetaldehyde is further oxidized to yield acetic acid, which is much less
volatile than acetaldehyde (the Henry’s law constant for acetic acid is 0.0133 Pa m3
mol1 compared to 7 Pa m3 mol1 for acetaldehyde, Niinemets and Reichstein
2003), although its emission has also been reported from flooded trees (Rottenberger
et al. 2008).
In general, the emission rates for ethanol are around one order of magnitude
higher compared to acetaldehyde emissions. In 24 h flooded Quercus robur trees,
for example, ethanol emission rates peaked at about 4.2 nmol m2 s1 , whereas
acetaldehyde emission amounted to only 0.3 nmol m2 s1 (Kreuzwieser 2002).
Similarly, in Alnus glutinosa, Q. rubra, Populus tremula and Q. ilex, ethanol
emission rates exceeded acetaldehyde release by factors of 5–10 (Holzinger et al.
2000; Copolovici and Niinemets 2010). Also, in non-woody species such as rice,
similar emission patterns have been observed (Boamfa et al. 2005; Mustroph
et al. 2006). However, some tree species from the Amazonian floodplain exhibit
somewhat different ethanol to acetaldehyde emission ratios (Parolin et al. 2004;
Rottenberger et al. 2008; Bracho-Nunez et al. 2012), with occasionally greater
acetaldehyde than ethanol emissions at some periods of flooding (e.g., Laetia
corymbulosa in Parolin et al. 2004). Unfortunately, there is not much data available
on the emissions of acetic acid as a consequence of flooding. In the work of
240 J. Kreuzwieser and H. Rennenberg

Rottenberger et al. (2008), flooded trees of L. corymbulosa emitted this organic acid
at slightly higher rates than non-flooded control trees. Compared to ethanol (up to
8.3 nmol m2 s1 ) or acetaldehyde (5 nmol m2 s1 ) release, acetic acid emission
rates were very low amounting to ca. 0.05 nmol m2 s1 in this species. In other
species studied, no such flooding effect was observed (Rottenberger et al. 2008).
The flux of a volatile compound from the leaf is determined by stomatal
conductance and the concentration gradient of the compound between substomatal
cavities and the atmosphere (Niinemets and Reichstein 2003; Harley 2013 in
this book). As stomatal conductances for ethanol, acetaldehyde and acetic acid
do not differ strongly (Niinemets and Reichstein 2003), differences in emission
rates must be mainly based on the concentration gradient which is determined by
the compound concentration in the liquid phase of the apoplast and its volatility,
reflected by the Henry’s law constant. Of the three C2 compounds, acetaldehyde
has the highest Henry’s law constant and therefore the highest volatility. Ethanol
is ca. 14-times less volatile than acetaldehyde and acetic acid even 3–4 orders
of magnitude less volatile than ethanol (depending on pH). Much higher ethanol
than acetaldehyde emission rates observed in many species therefore must be
caused by high ethanol concentrations in the apoplast as well as rapid acetaldehyde
consumption (Monson 2013 in this book). As ethanol in flooded trees is produced
in the roots, its leaf concentrations depend on the transport rate in the xylem.
On the other hand, the concentrations of acetaldehyde in the leaves of flooded
trees depend on (i) leaf ethanol abundance (Kreuzwieser et al. 2001), (ii) activity
of alcohol dehydrogenase which converts ethanol to acetaldehyde (Parolin et al.
2004; Ferner et al. 2012), and (iii) activity of aldehyde dehydrogenase, the enzyme
degrading acetaldehyde thereby producing acetic acid. Consequently, acetic acid
concentrations are determined by acetaldehyde abundance, activity of aldehyde
dehydrogenase (production) and the activity of acetic acid consuming enzymes.
Unfortunately, information on leaf apoplast concentrations of these volatiles is
scarce (ethanol) (Jaeger et al. 2009; Ferner et al. 2012) or even completely missing
(acetaldehyde, acetic acid). In leaves of non-flooded Fraxinus excelsior, Fagus
sylvatica and Q. robur, the ethanol concentrations were in a range of 50 g g1
FW to 200 g g1 FW and, thus, significantly higher than in the roots (Jaeger et
al. 2009; Ferner et al. 2012). The effects of flooding on leaf ethanol concentrations
seem to be inconsistent. Whereas leaf ethanol increased in F. sylvatica and Q. robur,
it was unaffected in F. excelsior and Fraxinus angustifolia (Jaeger et al. 2009; Ferner
et al. 2012).
Only a limited number of studies has investigated alcohol dehydrogenase activity
in leaves of flooded trees. Recently, it was demonstrated that there are no strong
species-specific differences in enzyme activity (Jaeger et al. 2009; Ferner et al.
2012). Nevertheless, similar to ethanol concentrations, in some species, alcohol
dehydrogenase activities in the leaves increased in response to flooding, but
remained constant in other species (Jaeger et al. 2009; Ferner et al. 2012). In addition
to the very similar alcohol dehydrogenase activity in different species, there was a
lack of any species-specific differences in the use of 14 C-labelled ethanol in the
foliar C metabolism; therefore, it was concluded, that acetaldehyde emission is
9 Flooding-Driven Emissions from Trees 241

Tilia cordata

Acetaldehyde emission (nmol m−2 s−1)

20 Acer platanoides
Quercus rubra

15

10

0
0 2 4 6
Xylem sap ethanol (mM)

Fig. 9.1 Correlation between xylem sap ethanol concentration and acetaldehyde emission in three
temperate deciduous species, Quercus rubra, Acer platanoides and Tilia cordata. Trees were
flooded for 0, 1, 2, 10 and 15 days (unpublished data, Kreuzwieser). At the end of each treatment
acetaldehyde emission rates were determined and xylem sap was extracted using the pressure
chamber technique (Scholander et al. 1965)

controlled by the amount of ethanol transported to the leaves (Ferner et al. 2012)
(Fig. 9.1). The good correlation of acetaldehyde emission rates with xylem sap
ethanol concentrations and ethanol abundance in leaves (Fig. 9.1) (Kreuzwieser
et al. 2001; Ferner et al. 2012) indicates that ethanol availability is the main
parameter determining the emission rates. This assumption is supported by the close
correlation of ethanol and acetaldehyde emissions from leaves of trees (Copolovici
and Niinemets 2010; Bracho-Nunez et al. 2012).

9.2.2 Relationships of Emissions of Fermentation Volatiles

with Species Flood Tolerance

Kreuzwieser et al. (1999) proposed that the production of ethanol in anoxia exposed
roots, its transport via the transpiration stream to the leaves and its oxidation and
re-use in the oxygen-exposed leaves of trees might be a physiological mechanism
of flood tolerance at the whole-plant level. It was further hypothesized that
acetaldehyde emission rates correlate with flood tolerance of trees (Kreuzwieser
et al. 2004). More recent results with moderately flood-tolerant Q. robur and flood-
sensitive F. sylvatica suggest that the metabolism in the leaves of at least these trees
species is less decisive for acetaldehyde emission and re-use of ethanol in oxidative
metabolism in the leaves (Ferner et al. 2012), and that rather the root processes
242 J. Kreuzwieser and H. Rennenberg

25

Acetaldehyde emission
20

(nmol m−2 s−1) 15

10

0
0 1 2 3 4
Flood tolerance

Fig. 9.2 Correlation of flood tolerance with acetaldehyde emission by flooded trees. Data are
mean ˙ SD values for Pinus sylvestris, Picea abies, Fagus sylvatica, Quercus robur, Q. rubra,
A. platanoides, Populus x canescens, P. tremula, Tilia cordata and Alnus glutinosa. Data are from
Copolovici and Niinemets (2010), Kreuzwieser et al. (1999) and Kreuzwieser and Rennenberg
(unpublished). Scoring of flood tolerance is according to Niinemets and Valladares (2006). Higher
values of the flood tolerance score indicate greater flood tolerance

determine the trees’ flood tolerance. The study of Copolovici and Niinemets (2010)
suggests that ethanol and acetaldehyde emission are inversely related to the species’
flood tolerance; for example, flood-sensitive Q. rubra emitted much more of these
volatiles than flood-tolerant P. tremula or A. glutinosa. Assuming that the ethanol
emitted by leaves was synthesized in the roots of the trees, it can be concluded
that trees’ flood tolerance is negatively associated with the amount of ethanol
produced in the roots, i.e., with root oxygen availability. However, flood tolerance
and ethanol production do not necessarily correlate (Raymond et al. 1985). In fact,
there is no general correlation between foliar acetaldehyde emissions the species
flood tolerance (Fig. 9.2).
Of course, the production of ethanol in the roots and its re-use in aerobic leaves
is only one physiological aspect of trees’ flood tolerance. This might be particularly
important for tree species not capable of extensive morphological acclimation
to flooding such as formation of adventitious roots and aerenchyma tissue. The
capacity for such morphological modifications that allow the plants to increase the
oxygen availability in the root zone, can be more strongly linked to species flood
tolerance (Drew 1997; Vartapetian and Jackson 1997). Such an increase of root
zone oxygen availability is expected to reduce the rate of fermentative processes,
thereby reducing ethanol and acetaldehyde emissions. This idea is supported by the
occurrence of aerenchyma rich adventitious roots in some Amazonian tree species
and subsequent reduced acetaldehyde and ethanol emission rates (De Simone et al.
2002a, b; Parolin et al. 2004).
9 Flooding-Driven Emissions from Trees 243

9.3 Physiology of Ethanol and Acetaldehyde Emission

9.3.1 Diurnal Variation Patterns: Influence of Stomata

Typically, acetaldehyde and ethanol are emitted from leaves of flooded trees at
high rates during the day and at low rates during night (Fig. 9.3). There is often
a peak of highest emissions in the early morning hours which decreases to lower
levels during the rest of the light-period. Such patterns are assumed to result
from daily patterns of stomatal conductance and consequently of transpiration
rates (Kreuzwieser et al. 2000; 2001). Especially the morning peak emission of
ethanol which has a low Henry’s law constant of 0.51 Pa m3 mol1 (Niinemets and
Reichstein 2003) can reflect stomatal control on emissions because a large pool of
ethanol in the liquid phase is rapidly released upon stomatal opening (Harley 2013
in this book; Niinemets and Reichstein 2003). In the case of acetaldehyde with a
somewhat higher Henry’s law constant of 7 Pa m3 mol1 (Niinemets and Reichstein
2003), the stomatal constraints are expected to pose a less strong effect on the
emissions. Application of abscisic acid to grey poplar (Populus x canescens) leaves
has clearly demonstrated that – similarly to isoprene (Fall and Monson 1992) –
stomatal opening does not play a dominant role in controlling acetaldehyde emission
rates (Kreuzwieser et al. 2001), although moderate temporal control by stomata
has been suggested by simulation analyses (Harley 2013 in this book; Niinemets
and Reichstein 2003). More importantly, the correlation between stomatal opening
and acetaldehyde emission suggests mainly an indirect control over acetaldehyde
emissions by determining the transpiration rates and thereby ethanol transport into
and abundance inside the leaf tissue. In fact, a significant linear correlation of

0.30 light dark light

acetaldehyde
ethanol
Acetaldehyde (nmol m−2 s−1)

0.25 6
Ethanol (nmol m−2 s−1)

0.20
4
0.15

0.10
2
Fig. 9.3 Daily pattern
of ethanol (squares) and 0.05
acetaldehyde (triangles)
emissions from flooded
Quercus robur trees 0.00 0
35 40 45 50 55
(Modified from
Kreuzwieser 2002) Time of Flooding (h)
244 J. Kreuzwieser and H. Rennenberg

acetaldehyde emission rates with xylem sap ethanol concentrations (Kreuzwieser

et al. 2000; Ferner et al. 2012) suggests that acetaldehyde emission is strongly
controlled by the abundance of ethanol in the leaf tissue. A steady transport of
ethanol from roots to the leaves is only ensured when transpiration takes place,
i.e., during the light period of a day. As discussed above, because acetaldehyde
is produced from the oxidation of ethanol by alcohol dehydrogenase, the activity
of this enzyme is also involved in determining the acetaldehyde emission rate.
Daily patterns of the alcohol dehydrogenase activity have, however, not been
demonstrated so far, although induction by ethanol has been demonstrated (Good
and Crosby 1989).

9.3.2 Effects of Flooding Duration

Besides the emission strength, the temporal pattern of elicitation of trace gas
emissions is strongly species-dependent. Some species, e.g., flood-tolerant Populus
x canescens or flood-sensitive Quercus rubra, react quickly with significantly
enhanced ethanol and acetaldehyde emission rates after some hours of oxygen
shortage in the roots (Kreuzwieser et al. 2004; Copolovici and Niinemets 2010).
This response can occur in a transient manner. In both species, the emission rates
drop after some days, reaching a steady emission level that is moderately higher
than in non-flooded controls. Other species react more slowly and show steadily
enhanced emission levels after some days of flooding (Kreuzwieser et al. 2004;
Parolin et al. 2004; Copolovici and Niinemets 2010) (Fig. 9.4). The driving forces
for such emission patterns remain unclear.
Apart from intrinsic differences among species, differences in soil conditions in
species habitat such as soil redox status development under anoxia (rate of reduction
of oxygen content of soil micropores and soil water by plant roots and aerobic
bacteria) may contribute to the observed emission patterns and apparent differences
between species. Soils with a lower portion of air-filled pores will be faster oxygen
depleted than soils entrapping more air. In natural ecosystems, the specific flooding
conditions are also of importance. It has been shown that stagnant flood water
promotes oxygen deficiency much stronger than a flowing water body (Kreuzwieser
et al. 2004). Since in none of the above mentioned studies soil characteristics were
investigated in detail, it cannot be excluded that differences in the establishment of
hypoxia/anoxia have contributed to the different emission patterns. Nevertheless, in
studies simultaneously investigating multiple species, same soils and same treatment
conditions have often been used, e.g., in the study of Copolovici and Niinemets
(2010), suggesting that detected patterns reflect intrinsic species responses.
As discussed in Sect. 9.2.2, differences between species in the rate of devel-
opment and level of expression of morphological adaptations such as aerenchyma
and adventitious root development may be involved in the drop of emissions
after prolonged flooding. Moreover, physiological features, such as a depletion of
9 Flooding-Driven Emissions from Trees 245

Fig. 9.4 Different temporal 9

patterns of acetaldehyde and Laetia corymbulosa
ethanol emissions through an 6 Acetaldehyde
extended flooding period in Ethanol
Amazonian trees (Modified 3
from Parolin et al. 2004)
0

Ethanol emission (nmol m−2 s−1)

Tabernaemontana juruana
4

0
Pouteria glomerata
1.5

1.0

0.5

0.0
0.3 Salix martiana

0.2

0.1

0.0
0 2 4 6 8 10
Flooding period (days)

carbohydrates, the substrate for alcoholic fermentation, may at least partially be

responsible. A reduction in fermentation-driven volatile emissions occurred at the
same time when a drop in plant sugar status was observed in flooded Fagus sylvatica
trees (Ferner et al. 2012).

9.4 Other Volatiles Released During Flooding

9.4.1 Emissions of Stress Volatiles

Besides the emission of fermentation-related volatiles ethanol, acetaldehyde and

acetic acid, the release of some other BVOCs is affected by flooding. A series of
publications have reported emissions of stress marker compounds in response to soil
flooding. This includes the emission of ethylene, wound BVOCs and the nitrogen-
containing volatile nitric oxide (Holzinger et al. 2000; Grichko and Glick 2001;
Agarwal and Grover 2006; Bracho Nunez et al. 2009; Copolovici and Niinemets
2010) (Fig. 9.5).
The release of some of these trace gases is clearly stimulated under flooding.
Such gases are either related to stress-induced injury of the leaves or might be a
246 J. Kreuzwieser and H. Rennenberg

Fig. 9.5 Temporal changes

LOX emission (nmol m−2 s−1) NO emission (nmol m−2 s−1)

in the emissions of NO and 1.2
volatile products of
lipoxygenase (LOX) reaction 0.8
from flooded Quercus rubra
and Alnus glutinosa trees 0.4 Quercus rubra
(Modified from Copolovici Alnus glutinosa
and Niinemets 2010). The
plant root systems were 0.12
completely submerged at day
1 and maintained in 0.06
waterlogged conditions
through the experiment 0.00
0.4

0.3

0.2

0.1

0.0
0 4 8 12 16 20
Flooding duration (days)

consequence of acclimation of the trees to flooding. Emission of volatile products

of the lipoxygenase pathway (Feussner and Wasternack 2002) (LOX products,
mainly C6 aldehydes) from leaves of flooded trees is certainly a stress indicator.
Consequently, emissions are higher from flood-sensitive plants than from more
tolerant species (Fig. 9.5; Copolovici and Niinemets 2010). LOX products are
derived from linoleic and linolenic acids released from cell membranes by the
action of phospholipases as a response to oxidative stress (Feussner and Wasternack
2002). The strength of LOX product emission is a marker of the severity of a
given stress factor (Beauchamp et al. 2005) and, in addition, of the sensitivity of a
species towards the particular stress (Copolovici and Niinemets 2010). The leaf NO
emission of flooded trees as observed by Copolovici and Niinemets (2010) might
also be an indicator of oxidative stress; similar to LOX product emissions it was
highest in flood-sensitive Q. rubra and lowest in flood-tolerant A. glutinosa.
In general, stress-induced NO emissions from leaves of plants are well docu-
mented; NO is involved in signal transduction pathways of biotic and abiotic stress
resistance (Delledonne et al. 1998; Mata and Lamattina 2001; Zhao et al. 2007,
2009; Xuan et al. 2010). The main pathway for NO production is most probably the
enzyme nitrate reductase, although there is also evidence of synthesis of NO by a
L-arginine-dependent nitric oxide synthase (NOS) (Besson-Bard et al. 2008; Corpas
et al. 2011).
9 Flooding-Driven Emissions from Trees 247

Fig. 9.6 Changes in isoprene a

50
emission (a) and net CO2 Quercus rubra

Isoprene emission
assimilation (b) rates in 40 Populus tremula

(nmol m−2 s−1)

Quercus rubra and Populus
tremula through the flooding 30
treatment (Modified from
Copolovici and Niinemets 20
2010, the same experiment as
in Fig. 9.5) 10

0
16 b

Net CO2 assimilation

(µmol m−2 s−1)
12

0
0 4 8 12 16 20
Flooding duration (days)

Stimulation of ethylene emission is a classic flooding response (Holzinger et al.

2000; Grichko and Glick 2001). Ethylene production and emission is closely
connected with the development of aerenchyma (for review see Evans 2003). In
aerenchyma formation, ethylene acts as a signal, inducing cell apoptosis and the
formation of air filled pores.
Release of methanol is also enhanced due to flooding (Copolovici and Ni-
inemets 2010). Methanol formation in plant tissues is closely related to cell wall
modifications. It is released as the result of the activity of cell wall associated
pectin methylesterases, which catalyze the demethylation of cell wall pectins during
relaxation and rigidification cycles during leaf expansion (Fall and Benson 1996;
Hüve et al. 2007; Ricard and Noat 1986), but also during degradation of cell walls
in maturing fruits and senescing leaves (Frenkel et al. 1998; Sun et al. 2012).
Thus, the formation of aerenchyma during flooding is closely connected to cell
wall degradation and the formation of intercellular air spaces, processes known to
stimulate methanol production (MacCann and Roberts 1991; Levy and Staehelin
1992; Nemecek-Marshall et al. 1995; Fall and Benson 1996).

9.4.2 Effects of Flooding on the Release of Constitutively

Emitted Volatiles

The emission of isoprene was reduced by waterlogging in the flood-tolerant Populus

tremula and flood-sensitive Quercus rubra, but this effect was much more pro-
nounced in the latter species (Copolovici and Niinemets 2010) (Fig. 9.6a). Reduced
248 J. Kreuzwieser and H. Rennenberg

isoprene emission was also found in flooded Garcinia macrophylla, an Amazonian

tree species (Bracho-Nunez et al. 2012). However, in the same study, other species
did not show any flood-induced changes in isoprene emission. Similarly, flooding
effects on monoterpene emission were inconsistent. The provenances of Hevea
spruceana derived from an igapó-type forest characterized by brownish, nutrient-
and sediment-poor flood water with low pH responded with a strong increase in
monoterpene emission (Bracho-Nunez et al. 2012). In contrast, the provenances
from a várzea-type forest characterized by nutrient and sediment rich “whitewater”
showed the opposite pattern (Bracho-Nunez et al. 2012). Similar to the igapó type
trees, flooded lemon (Citrus limon) trees also showed higher monoterpene emissions
than non-flooded control trees (Velikova et al. 2012). A function of isoprene in
quenching ROS in flooded plants has been discussed (Copolovici and Niinemets
2010), although there was no correlation of the capacity for constitutive isoprene
release and flood tolerance of a given species. The sharp decrease in isoprene
emissions from Q. rubra leaves due to flooding might be related to a general
disturbance of leaf metabolism, as can be concluded from strong reductions in
assimilation rates in this flood-sensitive species (Fig. 9.6b).

9.5 Conclusions

Flooding considerably affects the profile of volatile compounds emitted from

leaves of trees. While the emission of isoprene drops, reflecting disturbance of
photosynthetic processes, the release of products of glucose fermentation and
stress volatiles is stimulated. Ethanol is the key BVOC formed in the roots due
to fermentative processes in flooded trees and transported to the leaves via the
transpiration stream. In the leaves, ethanol is converted to acetaldehyde and acetic
acid, both of which can be emitted into the atmosphere in addition to ethanol.
The significance of these processes for ecosystem-scale acetaldehyde and ethanol
fluxes is still unclear. Most of the present data and conclusions are based on exper-
iments with tree seedlings investigated under more or less controlled conditions in
the greenhouse. The mechanisms of flooding-induced BVOC emission might be
of importance for ecosystems such as the Amazon forest where flooding regularly
occurs (Rottenberger et al. 2008). However, the fact that flooding-induced BVOC
emission rates usually drop during prolonged flooding, likely reflecting acclimation
suggests that the emissions are of limited significance for atmospheric processes
during the rest of the wet season. Other sources for these oxidized volatiles,
ethanol, acetaldehyde and acetic acid, such as e.g., photochemical production due to
oxidation or more reactive BVOCs, are assumed to be dominant compared to direct
emission by vegetation (Millet et al. 2010).
The typical stress volatiles induced in response to flooding are volatile products
of lipoxygenase (LOX) pathway (also called green leaf volatiles) and NO. There
9 Flooding-Driven Emissions from Trees 249

is evidence that flood-sensitive trees emit these compounds at higher rates than
tolerant species. In addition, ethylene and methanol can also be released in response
to flooding. The site of release of the volatiles is currently unclear, but it is likely
that ethylene and methanol are produced both in the roots and leaves. Ethylene
stimulates the formation of aerenchyma, a morphological adaptation of trees to
flooding that improves the supply of the root system with atmospheric oxygen
during the flooding period, while methanol is released from cell wall pectins during
aerenchyma formation.

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Chapter 10
Modification of BVOC Emissions by Changes
in Atmospheric [CO2 ] and Air Pollution

Carlo Calfapietra, Emanuele Pallozzi, Ilaria Lusini, and Violeta Velikova

Abstract Biogenic volatile organic compounds (BVOCs) produced by trees partic-

ipate in the formation of air pollutants such as ozone and particulate matter. At the
same time, the metabolic processes responsible for these emissions are sensitive
to ozone and other air pollutants, as well as the solar radiation flux, which is
affected by atmospheric particulate concentration. Recent anthropogenic increases
in the atmospheric carbon dioxide concentration are also capable of affecting BVOC
emissions, although the mechanisms behind these responses can produce variable
effects depending on the plant species. Mechanisms of air pollutant effects on
BVOC emissions are reviewed and dose-response relationships across a variety of
trees with differing pollutant tolerance and emission capacity are compared. From
this broad analysis, generalized response patterns have been developed. This chapter
emphasizes the need to consider the interactions between BVOC emissions and
ozone to understand plant behaviour in future climates.

C. Calfapietra () • E. Pallozzi

Institute of Agro-Environmental and Forest Biology (IBAF), Consiglio Nazionale delle Ricerche
(CNR), Via Marconi 2, 05010 Porano, TR, Italy
e-mail: [email protected]
I. Lusini
Institute of Agro-Environmental and Forest Biology (IBAF), Consiglio Nazionale delle Ricerche
(CNR), Via Marconi 2, 05010 Porano, TR, Italy
Department for Innovation in Biological, Agro-Food and Forest Systems, University of Tuscia,
Via San Camillo de Lellis s.n.c, 01100 Viterbo, Italy
V. Velikova
Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev
Str., Bl. 21, 1113 Sofia, Bulgaria

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 253
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 10,
© Springer ScienceCBusiness Media Dordrecht 2013
254 C. Calfapietra et al.

10.1 Introduction

10.1.1 Global Change Projections

It is widely acknowledged that the atmosphere has been affected by human

activities, beginning from the Industrial Revolution and continuing to the present.
Human-induced increases in the emissions of greenhouse gases are likely re-
sponsible for an increase in global average temperature, altering the hydrological
cycle, with more intense droughts and floods (IPCC 2007). Anthropogenic carbon
dioxide (CO2 ) emission is the most important forcing variable affecting changes
in climate over the past century. CO2 concentration in the atmosphere remained
relatively constant in the range of 260–280 mol mol1 over long periods before
the Industrial Revolution, when it has continuously increased from approximately
270 mol mol1 in the late nineteenth century to a current atmospheric average
of approximately 400 mol mol1 (Keeling et al. 2009). Projections from global
carbon cycle models show that concentrations of atmospheric [CO2 ] could increase
to 500–1,000 mol mol1 by the year 2100 (IPCC 2007). The main cause of rising
CO2 emissions is burning of fossil fuels (coal, oil and natural gas), but emissions are
also high due to deforestation and subsequent biomass burning and due to cement
manufacturing.
Other greenhouse gases produced by human activities contribute to radiative
forcing. Production of tropospheric ozone (O3 ) is the third most important contribu-
tor to anthropogenic radiative forcing after emissions of carbon dioxide and methane
(CH4 ). Ozone is produced in the troposphere by nitrogen oxides (NOx ) interacting
with non-methane volatile hydrocarbons (NMVOCs) in the presence of blue light in
the visible spectrum. Background tropospheric O3 concentrations continue to rise
due to human activities (IPCC 2007), increasing from pre-industrial concentrations
of less than 10 nmol mol1 (ppb) (Volz and Kley 1988) to average summertime
concentrations of 40 nmol mol1 (Fowler et al. 1999). Ozone concentrations in the
troposphere have been monitored by the environmental agencies of several countries
because it, as a highly reactive oxidizing agent, is a principal component of urban
and suburban air pollution.

10.1.2 Role of BVOC Emissions in Changing the Atmosphere

Biogenic volatile organic compounds (BVOCs) are released by vegetation to the

atmosphere in significant quantities accounting for up to 5–10 % and more of total
net carbon exchange into the atmosphere (Peñuelas and Llusià 2003). They can
strongly contribute to the global change and play a crucial role in atmospheric
composition because of their reactivity. For isoprene, this amount is on the order
of 400–500 Tg C year1 (Archibald et al. 2011). Many studies have demonstrated
that biogenic volatile organic compounds (BVOCs) contribute to the formation of
tropospheric ozone (Chameides et al. 1988; Tao et al. 2003; Bell and Ellis 2004;
10 Modification of BVOC Emissions by Changes in Atmospheric [CO2 ]. . . 255

Hogrefe et al. 2011) in the presence of NOx . The release of BVOCs also constitutes
a significant input of precursors for photochemical oxidants (Simpson et al. 1995;
Trainer et al. 1993). Most recently, evidence has been provided that the emission
of BVOCs from tropical forests, particularly isoprene, plays an important role
in recycling hydroxyl radicals, implying that BVOC emissions occupy a central
role in buffering the oxidative capacity of the atmosphere (Lelieveld et al. 2008;
Taraborrelli et al. 2012). Thus, studies of the relation of BVOC emissions to
increases in atmospheric O3 concentrations are likely to reveal key processes that
control the oxidative capacity of the troposphere and control important surface-
atmosphere feedback loops with implications for climate change (Ashworth et
al. 2013; Kulmala et al. 2013 in this volume). Changing forest management
practices lead to further important implications. Millions of hectares of fast-growing
plantations are being planted across the world for biomass production and with the
aim to sequester large amount of atmospheric CO2 . Most of the species used are
poplars (Populus spp.), willows (Salix sp.) and eucalypts (Eucalyptus spp.) that are
strong BVOC emitters (Owen et al. 2013). This means a large BVOC load into
the atmosphere, especially in developing areas of the planet where growing levels
of NOx are being emitted, thus exacerbating the pollution potential in these areas
(Hewitt et al. 2009). These aspects recall the need to reconsider the bio-engineering
for the future, considering that we are now able to clone the isoprene/monoterpene
synthase gene from many species and to create transgenic lines of several species
which do not produce and emit BVOCs (Miller et al. 2001; Behnke et al. 2011).
In this chapter, we will focus on the effects of atmospheric [CO2 ] and [O3 ] on
BVOC emission at the leaf scale and these effects will be considered within the
broader context of climate change (Fig. 10.1). We first analyse the experimental
setups and methodologies in studies of [CO2 ] and [O3 ] effects. Then we analyse
the leaf-level responses to [CO2 ] and [O3 ] and combinations of both, and finally
investigate the opportunities of how the reported effects can be best included in
models. We separate between instantaneous effects that result from biochemical
responses (Li and Sharkey 2013 for detailed description of overall emission mecha-
nisms) and acclimation effects that reflect modifications in enzymatic capacities for
BVOC synthesis (Monson 2013 for the overall philosophy of separating the BVOC
flux controls).

10.2 Rationale and Methodological Issues of BVOC Studies

Under Elevated [CO2 ] and [O3 ]

10.2.1 Experiments in Controlled Conditions

The responses of BVOC emissions to changes in atmospheric composition have

been investigated with different methods both in the laboratory controlled conditions
and in the field, manipulating or focusing on natural gradients of one or more factors.
However, the response of plant emissions can vary considerably depending on the
256 C. Calfapietra et al.

Fig. 10.1 Direct and indirect effects of increased CO2 concentration at leaf and ecosystem levels
on BVOC emissions. [CO2 ] may directly stimulate (upwards arrows) or inhibit (downwards
arrows) the emissions, and the [CO2 ] effects may further indirectly be altered by [CO2 ] effects
on temperature

specific BVOC emitted, the plant species involved, the scale of interest (single
leaf vs. whole plant) and the time of interest (short-term vs. long-term responses).
Several laboratory studies have been conducted on the effects of high concentrations
of CO2 and/or O3 , where concentrations could be controlled artificially and changed
quickly according to experimental design. Experiments with rapid changes in CO2
concentrations have been originally coupled with studies of photosynthesis (Monson
and Fall 1989) and provided important insights regarding the regulatory processes
behind the formation and emission of BVOCs. These experiments are usually
carried out at the leaf level and are often limited to a temporal scale that is in a
range of few hours or some days. These instantaneous responses are different from
those observed in leaves grown for a long period under elevated [CO2 ] or elevated
[O3 ] because of acclimation mechanisms which can occur over time (Fig. 10.1,
Monson 2013 in this volume). Both relatively rapid and longer-term experimental
manipulations can be conducted by exposing entire plant or entire ecosystems to
elevated concentrations of CO2 and/or O3 , because in these cases, the regulations
can occur at different temporal and spatial scales (Niinemets 2012 for a review).
This is due mainly to canopy effects on microclimatic conditions, which, in turn,
can influence BVOC emission directly and indirectly.
10 Modification of BVOC Emissions by Changes in Atmospheric [CO2 ]. . . 257

10.2.2 Localized Fumigation (LOF) System Experiments

The single leaf laboratory fumigation experiments provide insight into the phys-
iological and biochemical changes induced by acute and short-term exposure to
elevated CO2 and/or O3 concentrations. However, the results observed with these
laboratory fumigation systems are not necessarily transferable to field conditions.
The localized CO2 and O3 fumigation (LOF) system is a promising compromise
between highly controlled laboratory experiments and experiments under field
conditions (Pinelli and Tricoli 2004; Brilli et al. 2007; Loreto et al. 2007). LOF
systems provide short-term fumigation of individual leaves on field-grown or potted
plants, which have developed under the normal background atmospheric CO2 or O3
concentration. The LOF system enables investigation of physiological responses of
leaves with different age on the same plant and exposed to a broad range of CO2 or
O3 concentrations. This enables researchers to continue to control for other factors
determining plant response to fumigation, such as genetic variability, meteorological
dynamics and soil factors (Pinelli and Tricoli 2004).

10.2.3 Field Experiments

Studies focusing on BVOC emissions under different atmospheric compositions in

the field are often biased by the diverse experimental conditions used in the different
studies. Plants growing at different CO2 and/or O3 concentrations can exhibit
different within-canopy light and/or temperature gradients due to the effects of
seasonal changes in leaf area profiles and changes in soil moisture, which can affect
canopy latent heat exchange (Gielen et al. 2003; Liberloo et al. 2007). Thus, in field
studies, it is particularly important to measure BVOC emission rates for individual
leaves under standard light and temperature (30 ı C and 1,000 mol m2 s1 )
conditions, and study within-canopy variation in foliage BVOC emission capacity
(Centritto et al. 2004; Niinemets et al. 2010b).
When experiments are performed in the field, generally only one or two factors
can be manipulated, while laboratory conditions allow researchers to impose
multiple factors and their combinations when needed and produce observations
under comparable conditions. Experimental manipulation of pollutant concentration
at the scale of several hectares or an entire region is impossible with current
technology and resources. It is possible, however, to perform experiments on the
effects of changed atmospheric composition on isolated trees or stands of trees in
natural environments. In addition, studies of O3 pollution in natural environments
can be carried out using both natural (Winner et al. 1989) and regional gradients
(Karnosky et al. 1999; Arbaugh et al. 2003). Unfortunately, the concentration of
O3 across the gradient is not constant, and the presence of gradients in other
environmental factors could confound the O3 effects (Paoletti et al. 2005).
In the last 20 years, the free-air CO2 enrichment (FACE) technique has allowed
researchers to fumigate portions of ecosystems in field condition, thus maintaining
258 C. Calfapietra et al.

control over other environmental variables and avoid the limitations of the open-top
chambers (OTC) where alteration of microclimatic characteristics often occurred.
This technique has been applied also to [O3 ] (Karnosky et al. 1999; Dickson
et al. 2000).
An important factor when working with air pollutants in free-air exchange
experiments, and particularly when working with O3 , is the high reactivity between
many BVOCs and O3 causing the reduction of BVOC concentrations within the
treatment rings. This effect may be especially important in the most polluted
treatments due to BVOCs and O3 and BVOCs and other, secondary oxidants (Fares
et al. 2010; Jardine et al. 2012). Thus, when leaves are isolated for study in these
experiments, and leaf-level measurements are performed, leaves should be measured
in the presence of clean air flows to gain insight into BVOC emission potentials
(Loreto and Velikova 2001; Brilli et al. 2011; de Gouw and Warneke 2007).

10.2.4 Natural CO2 Spring Experiments

Natural CO2 springs provide an opportunity to study the effects of elevated CO2
concentrations on BVOC emissions, together with other related parameters, in trees
or stands of trees growing near the CO2 source. A limitation of these studies can
be the frequent fluctuation of [CO2 ] that can compromise the establishment of
a “target” concentration at any particular distance from the CO2 source, and the
lack of control plots at ambient [CO2 ] with similar characteristics of soil, water,
and nutrient availability and with similar plant species, and often also with similar
light level. In addition to difficulties with appropriate control treatment, another key
shortcoming of CO2 spring experiments is the lack of replication. Moreover, the
presence of pollutants in several of these CO2 springs, mostly sulphur-containing
compounds such as H2 S and SO2 , can make it difficult to isolate the CO2 effect on
BVOC emission and can compromise the measurement by increasing background
contamination and the reactivity with BVOCs (Paoletti et al. 2005).

10.3 Effects of Elevated Atmospheric [CO2 ] on Plant

BVOC Emissions

The effect of the progressive rise of atmospheric CO2 concentration on the emission
rate of BVOCs has been investigated in a number of studies, and different responses
have often been observed (Table 10.1, Fig. 10.2). The responses of plant emissions
to rising [CO2 ] can differ among species and among plants of different ages. The
responses can also vary with different experimental lay-outs, times of exposure,
and with water and nutrient supply, thus complicating the comparison of the results
10 Modification of BVOC Emissions by Changes in Atmospheric [CO2 ]. . . 259

Table 10.1 Variability in BVOC emission under changing CO2 concentrations in different plant
species and different fumigation experiments
Species Experimental system Effect Reference
Isoprene emission
Short-term experiments
Populus tremuloides Leaf cuvette  Monson and Fall (1989)
Quercus rubra Leaf cuvette  Loreto and Sharkey (1990)
Quercus pubescens Natural CO2 springs  Rapparini et al. (2004)
Long-term experiments
Populus deltoides Biosphere 2 facility  Rosenstiel et al. (2003)
Populus x euramericana Free-air CO2 enrichment a Centritto et al. (2004)
(FACE)
Populus deltoides Biosphere 2 facility Db Pegoraro et al. (2004)
Quercus robur Well-ventilated  Possell et al. (2004)
greenhouse
Quercus pubescens Natural CO2 springs D Rapparini et al. (2004)
Phragmites australis Natural CO2 springs  Scholefield et al. (2004)
Mucuna pruriens Whole-plant chambers (C)c Possell et al. (2005)
Populus tremuloides Free-air CO2 enrichment D Calfapietra et al. (2007)
(FACE)
Populus alba Free-air CO2 enrichment D Loreto et al. (2007)
(FACE)/Laboratory
experiment
Liquidambar styraciflua, Free-air CO2 enrichment  Monson et al. (2007)
Populus tremuloides
Populus deltoides Biosphere 2 facility Dd Pegoraro et al. (2007)
Populus tremuloides Free-air CO2 enrichment e Calfapietra et al. (2008)
(FACE)
Eucalyptus globulus, Controlled-environment  Wilkinson et al. (2009)
Liquidambar growth chambers
styraciflua, Populus
deltoides, Populus
tremuloides
Ginkgo biloba Open-top chambers C Li et al. (2009)
Acacia nigrescens Controlled-environment  Possell and Hewitt (2011)
growth chambers
Populus tremula x P. Open-top chambers D Sun et al. (2012)
tremuloides
Monoterpene emission
Short-term experiments
Quercus ilex Natural CO2 springs  Rapparini et al. (2004)
Long-term experiments
Pinus ponderosa Open/top chambers and D Constable et al. (1999)
controlled-
environment
Terracosm
(continued)
260 C. Calfapietra et al.

Table 10.1 (continued)

Species Experiment Effect Reference
Pseudotsuga menziesii
Quercus ilex Open-top chambers , Cf Loreto et al. (2001)
Quercus ilex Controlled-environment C Staudt et al. (2001)
greenhouse
Adenostoma fasciculatum Biological field station D Baraldi et al. (2004)
Ceanothus greggii
Quercus ilex Natural CO2 springs D Rapparini et al. (2004)
Betula pendula Open-top chambers D Vuorinen et al. (2005)
Pinus sylvestris Closed-top C Räsänen et al. (2008)
environmental
chambers
Ginkgo biloba, Taxodium Growth chambers C,, Dg Llorens et al. (2009)
distichum
Metasequoia
glyptostroboides
Sequoia sempervirens,
Nothofagus cunninghamii
The treatment time varied between 3 h and 10 days for short-term and from 30 days to 8 years for
long-term treatments
a
Decrease age-dependent
b
Drought and high vapour pressure deficit (VPD) offset the inhibitory effect of elevated CO2
c
Increase in sub-ambient atmospheric [CO2 ]
d
Plants grown at elevated [CO2 ] were less sensitive to drought stress
e
Decrease mainly in the O3 -sensitive clone
f
Only limonene emission increased
g
Species-specific response

reported in the literature. Here we analyse the patterns for key BVOCs, isoprene
and monoterpenes, and address the possible sources of variation in experimental
observations.

10.3.1 Effects on Isoprene Emissions

10.3.1.1 General Patterns

There is a high degree of species-specific variability in the response of isoprene

emission to elevated [CO2 ], although generally a decreased emission is observed
both in the short-term and in the long-term experiments (Fig. 10.2). Here we
summarize a few responses in several case studies to emphasize the general trends,
but also highlight exceptions.
Different species of Populus have exhibited a certain variability in responses
to increasing [CO2 ], although an inhibition effect prevailed in most of the
studies. Decreased emissions were observed in the hybrid poplar Populus x
10 Modification of BVOC Emissions by Changes in Atmospheric [CO2 ]. . . 261

Fig. 10.2 Effects of elevated CO2 , O3 and CO2 C O3 concentrations on isoprene and monoterpene
emissions. Bars represent mean (˙SE) values derived from studies in Tables 10.1 and 10.2.
Included are only studies where enough quantitative information was available to derive estimates
for different treatments. In studies with multiple additional treatments such as drought or warming,
only the control treatments were used. In studies with multiple CO2 and O3 treatments or species,
a mean value for each study was used. Across the included treatments, the ranges of [CO2 ] were
550–1,200 mol mol1 (ppmv) for elevated [CO2 ], and the applied [O3 ] concentrations were
75–300 nmol mol1 (ppbv) for elevated [O3 ]. The treatment time varied between 3 h and 10 days
for short-term and from 30 days to 8 years for long-term treatments. The numbers next to the bars
denote the number of studies

euramericana (Centritto et al. 2004) and in P. deltoides (Rosenstiel et al. 2003;

Wilkinson et al. 2009). The decreased emissions in P. deltoides were not observed
when the plants were exposed to drought stress (Pegoraro et al. 2004), possibly
due to decreased intercellular CO2 concentrations, which likely counteracted the
influence of elevated atmospheric [CO2 ] due to reductions in stomatal conductance
(Sects. 10.3.1.2 and 10.3.2). Significant decreases in isoprene emission in quaking
aspen (Populus tremuloides) have also been observed (Wilkinson et al. 2009),
especially in one clone (Calfapietra et al. 2008). Monson et al. (2007) demonstrated
evidence of an active down-regulation of isoprene emissions during long-term
growth in FACE experiments with sweetgum (Liquidambar styraciflua) forest
in Tennessee, USA and P. tremuloides stands in Wisconsin, USA. However, no
significant effect of elevated [CO2 ] on isoprene emission was observed in an
experiment with white poplar (Populus alba) (Loreto et al. 2007), and in hybrid
aspen (Populus tremula x P. tremuloides) (Sun et al. 2012). Furthermore, in the
study of Sharkey et al. (1991), elevated [CO2 ] resulted in increased isoprene
emissions in white oak (Quercus alba) and inhibited emissions in P. tremuloides.
In gymnosperm Gingko biloba, Li et al. (2009) showed an increase of isoprene
emission under elevated [CO2 ].
262 C. Calfapietra et al.

10.3.1.2 Drought, N Availability, Temperature and Ontogeny Effects

Of particular interest for the responses of isoprene emissions to changes in [CO2 ] is

the effect of drought, which can offset the negative effect of elevated [CO2 ] (Loreto
et al. 2001; Rapparini et al. 2004; Pegoraro et al. 2004, 2007). Results from Populus
deltoides in Biosphere 2 facility clearly showed this effect (Pegoraro et al. 2007).
Emissions from poplar trees grown under elevated [CO2 ] showed a significant
stimulation under drought as compared to those grown under ambient [CO2 ].
Only when drought stress became severe, the emission decreased under elevated
[CO2 ]. Increases in isoprene emissions under elevated [CO2 ] and drought are likely
due to decreases in leaf stomatal conductance (gs) and decreases in intercellular
CO2 concentration (Ci ) that alleviated the inhibitory effect of elevated [CO2 ]
(Sect. 10.3.2). These results were consistent with studies in open-top chambers by
Loreto et al. (2001).
It is not yet conclusively clear how nitrogen availability alters isoprene emissions
under ambient [CO2 ], but nitrogen availability can modify the CO2 -response of
isoprene emission. Possell et al. (2004) showed that the addition of nutrients
reduced, but did not completely cancel, the negative impact of elevated [CO2 ] on
isoprene emission in Quercus robur. Overall, study-to-study variations in elevated
[CO2 ] effects on isoprene emission (Sect. 10.3.1.1) have been attributed to variations
in plant nutrition among the treatments, with studies demonstrating inhibition being
possibly characterized by more sever nutrient limitations under elevated [CO2 ]
(Sun et al. 2012).
Generally, elevated temperatures stimulate isoprenoid emission (Monson and
Fall 1989; Monson et al. 1994; Sharkey et al. 1999; Petron et al. 2001) so it
is likely that the inhibitory effect of elevated [CO2 ] can be counteracted by the
higher temperatures that is predicted in future high [CO2 ] environments (Rapparini
et al. 2004). In fact, at high temperature, the rate of isoprene emission has been
shown to became less sensitive to [CO2 ] (Loreto and Sharkey 1990; Rasulov
et al. 2010). Observations on the effects of the stress factors on isoprene emission
support the hypothesis that other environmental stresses, such as drought and high
temperature, particularly important in the Mediterranean area, can increase the
isoprene emissions and offset the negative effect of elevated [CO2 ] (Rapparini
et al. 2004).
Centritto et al. (2004) studying P. x euramericana saplings growing in a FACE
experiment showed that isoprene emission was age-dependent and decreased under
elevated [CO2 ]. In fact, only fully-mature leaves emitted isoprene, while young
developing leaves did not emit isoprene. Leaf ontogeny was accelerated by growth
at elevated [CO2 ], although the leaf plastochron index for reaching the maximum
isoprene emission rate and its decline in aging leaves remained unaltered under
elevated and ambient [CO2 ]. There is evidence of enhanced leaf area formation
under elevated [CO2 ], but the stimulation of total leaf area by elevated [CO2 ] is
often a transient effect and can disappear after the first years of growth (Saxe
et al. 1998; Norby et al. 1999; Gielen and Ceulemans 2001; Nowak et al. 2004).
Thus, provided total leaf area remains similar, the integrated whole-plant isoprene
10 Modification of BVOC Emissions by Changes in Atmospheric [CO2 ]. . . 263

emission is expected to be significantly lower under elevated [CO2 ] as has been

observed by Loreto et al. (2007). However, there is evidence that leaf area can
increase under elevated [CO2 ], resulting in enhanced whole plant isoprene emissions
(Sun et al. 2013).

10.3.1.3 Sub-ambient [CO2 ] Responses

Isoprene emissions have been shown to be enhanced in many species under sub-
ambient CO2 concentrations similar to those during the Last Glacial Maximum
(about 180 mol mol1 ). Such an increase has been reported in Eucalyptus globulus
(Wilkinson et al. 2009), Mucuna pruriens and Arundo donax (Possell et al. 2005).
Possell et al. (2005) suggested that during the glacial period the emissions were
still lower due to cooler temperatures, and possibly lower leaf area index (LAI). In
fact, Way et al. (2011) using transgenic poplar (P. x canescens) with suppressed
isoprene emissions suggested that isoprene biosynthesis may have evolved under
low atmospheric CO2 concentrations. They grow emitting and non-emitting trees
under low (190 mol mol1 ) and high (590 mol mol1 ) CO2 concentrations and
tested the effects of short, transient periods of high light and temperature. Isoprene-
emitting plants grown under low [CO2 ] emitted twice as much isoprene as those
grown under high [CO2 ] and had significantly greater tolerance of sunflecks than
non-emitting trees (Way et al. 2011). Thus, this evidence suggests that rising [CO2 ]
might reduce the functional benefits of isoprene in the future (see also the chapter
of Fineschi et al. 2013).

10.3.2 Mechanisms of [CO2 ] Responses of Isoprene Emission

There is still a debate about the mechanism behind the decrease of isoprene
emissions under elevated [CO2 ]. Rosenstiel et al. (2003) provided evidence of
a clear reduction of isoprene emission with the rise of CO2 concentration, at
the protoplast, leaf and ecosystem levels, despite the increase of biomass and
photosynthetic rates under elevated [CO2 ]. There was a positive correlation between
the rate of isoprene production and the cellular concentration of dimethylallyl
diphosphate (DMADP), the immediate precursor of isoprene biosynthesis. Such a
reduction of DMADP pool size by high [CO2 ] has been further shown in several
other studies (Rasulov et al. 2009; Possell and Hewitt 2011; Sun et al. 2012). Thus,
the reduction of DMADP production can constitute the main reason for suppression
of isoprene emission under elevated [CO2 ]. However, in the reported patterns, it is
important to distinguish between instantaneous and acclimation responses. Isoprene
emission rate essentially instantaneously decreases after rising the [CO2 ] above
ambient level (Rapparini et al. 2004; Scholefield et al. 2004) (Loreto and Sharkey
1990; Rasulov et al. 2009; Sun et al. 2012). Thus, when measured under given
growth [CO2 ] environment, i.e., ambient-[CO2]-grown plants are measured under
264 C. Calfapietra et al.

ambient [CO2 ], and elevated-[CO2]-grown plants are measured under elevated

[CO2 ], the response to elevated [CO2 ] consists of two processes, immediate, short-
term response to differences in CO2 concentration and longer-term acclimation
effect to growth [CO2 ] (Sun et al. 2012; Monson 2013), and here we separately
discuss both processes.

10.3.2.1 Instantaneous [CO2 ] Response

DMADP formation via chloroplastic 2-C-methyl-D-erythritol 4-phosphate/1-

deoxy-D-xylulose 5-phosphate pathway (MEP/DOXP pathway) starts with
condensation of glyceraldehyde 3-phosphate and pyruvate. The origin of
chloroplastic pyruvate is not fully clear, but it has been postulated that a significant
fraction of carbon entering the chloroplastic DMADP pool might originate from
cytosolic phosphoenol pyruvate (PEP) (Monson et al. 2009; Wilkinson et al. 2009).
PEP, derived from cytosolic glycolysis, is the substrate for a number of cellular
metabolic pathways and for mitochondrial respiration. PEP enters into the Krebs
cycle after conversion to oxaloacetic acid by the cytosolic enzyme PEP carboxylase
(PEPc). Rosenstiel et al. (2003) hypothesized that the formation of isoprene depends
on the transport of PEP into the chloroplast from the cytosol. As inhibition of
cytosolic PEPc seemingly caused an increase in isoprene emissions under elevated
[CO2 ], they postulated a metabolic competition for PEP between PEP carboxylase
and its transport into the chloroplast (Rosenstiel et al. 2003). These experiments
were used to hypothesize that reduction of isoprene emission under elevated [CO2 ]
is due to reduced DMADP concentrations in the chloroplast due to competition
between PEP carboxylase and chloroplast uptake.
Additional pieces of evidence have provided support to the postulated mecha-
nism. Loreto et al. (2007) observed a positive, although weak, correlation between
dark respiration and isoprene emission rates in leaves exposed to or grown under
different [CO2 ] both in the field and laboratory experiments. At the same time, a
negative correlation was observed between the activity of PEPc and the isoprene
emission rate (Loreto et al. 2007). Thus, they provided further evidence for the role
of competition for PEP on isoprene emissions, but ruled out a role of mitochondrial
respiration as part of this competition. Using 13 CO2 -labelling, Trowbridge et al.
(2012) observed in P. x canescens plants grown under sub-ambient [CO2 ] a greater
incorporation of “older” non-labeled carbon in isoprene than under high [CO2 ], and
demonstrated that the older carbon comes from pyruvate substrate. They suggested
that this older carbon in pyruvate is most likely of cytosolic origin (Trowbridge
et al. 2012).
An alternative hypothesis to explain the inhibitory effect of high CO2 concen-
tration on isoprene emission is the regulation of isoprene synthesis pathway by
the availability of ATP and NADPH (Rasulov et al. 2009). There is a positive
correlation between ATP level and isoprene emission (Loreto and Sharkey 1993),
and the pool size of ATP is lower under higher than under ambient [CO2 ] (Cardon
and Berry 1992; Delwiche and Sharkey 1993). Furthermore, Rasulov et al. (2009)
10 Modification of BVOC Emissions by Changes in Atmospheric [CO2 ]. . . 265

demonstrated that isoprene emission was inhibited by both high and very low CO2
concentration (below ca. 100–180 mol mol1 ). Both are conditions under which
ATP formation is inhibited (Kiirats et al. 2009). This reduction of isoprene emission
by low CO2 concentration cannot be explained by PEP transport. We note that both
PEPc and ATP hypotheses of CO2 effects on isoprene emission agree in that the
inhibition results from reduced DMADP pool size, but the debate is over what
causes DMADP pool size to decrease under high [CO2 ].

10.3.2.2 Acclimation Response

Nowadays, acclimation of isoprene emission capacity to increased CO2 concentra-

tions is considered as a key gap in understanding biochemistry of isoprene formation
(Sharkey 2009) and identification of possible adaptive modifications is difficult.
Available data on these adaptive changes are contrasting. Some studies have been
carried out in the vicinity of natural CO2 springs which provided access to the long-
term exposure to elevated [CO2 ] and the onset of possible adaptation mechanisms
(Rapparini et al. 2004; Scholefield et al. 2004). Scholefield et al. (2004) studied
Phragmites australis grown in the vicinity of natural CO2 springs along three
streams of freshwater at the bottom of the crater. The basal isoprene emission rate
was higher in plants grown at ambient [CO2 ] (control site) than in those grown
under elevated [CO2 ], but the inhibition under elevated [CO2 ] was attenuated when
expressed on a leaf nitrogen content basis. It was also evident that the inhibition
of isoprene emission due to high CO2 concentration followed the gradient of CO2
concentration from the bottom to the edge of the CO2 vent. In particular, isoprene
synthase activity was strongly reduced at the bottom of the springs (where CO2
concentration is around 900–1,100 mol mol1 ) than in the peripheral stand (CO2
concentration around 700 mol mol1 ). However, no inhibition at the branch-
level isoprene emissions was observed in Q. pubescens grown in natural CO2
springs (Rapparini et al. 2004). The higher leaf temperature at elevated [CO2 ] in
natural springs and the interaction with multiple stresses as recurrent drought may
compensate the negative effect of long-term exposure to high concentrations of
CO2 . The same findings have been obtained in a recent study of Sun et al. (2012)
which showed no significant differences in isoprene emission rate among ambient-
and elevated-[CO2] grown plants, and in fact enhanced emissions from elevated-
[CO2 ] grown plants when studied at the same ambient CO2 concentration. In this
study, isoprene emission rate was enhanced at higher [CO2 ] under saturating light
conditions.
Changes in isoprene emission capacity in plants developed under different [CO2 ]
may reflect changes in isoprene synthase activity, and/or changes in the precursor
DMADP pool size. Calfapietra et al. (2007) studying the response of field-grown
quaking aspen (P. tremuloides) trees exposed to elevated [CO2 ] observed a non-
significant decrease of isoprene synthase (IspS) protein levels under elevated [CO2 ].
However a significant inhibitory effect of elevated [CO2 ] on isoprene emission rates
was observed as a result of decreased DMADP levels (Calfapietra et al. 2008).
266 C. Calfapietra et al.

Analogous patterns were reported for hybrid aspen (P. tremula x P. tremuloides)
by Sun et al. (2012). In Possell and Hewitt (2011), elevated [CO2 ]-driven inhibition
of isoprene emissions in the tropical African tree Acacia nigrescens was associated
with both reduced DMADP pool size and isoprene synthase activity. Clearly, there
are study-to-study differences in changes in isoprene synthase activity and DMADP
pool size in plants acclimated to elevated [CO2 ], and future work should focus on
resolving this variability.

10.3.3 Influence of Elevated [CO2 ] on Monoterpene Emissions

In understanding monoterpene emission responses to elevated [CO2 ], it is critical to

distinguish among storage emissions that are prevailing in species with specialized
storage structures and emissions from immediate synthesis that are dominating the
emissions in species lacking specialized storage (Grote et al. 2013 for a discussion).
In the case of storage emissions, elevated [CO2 ] is expected to alter the size of
storage pool and accordingly, the effects can be explained on the basis of possible
alterations of sink-source balance (Constable et al. 1999; Litvak et al. 2002). In
contrast, the response of immediate emissions in species lacking storage tissues
may approximate the elevated [CO2 ] effects on isoprene emissions (Niinemets et al.
2010a). Thus, differences among species in growth [CO2 ] effects on monoterpene
emissions can be accounted for by differences in the emission mechanisms, tissue
storage vs. immediate emission.
Monoterpene-storing conifer species such as Pinus ponderosa and Pseudotsuga
menziesii showed no differences in tissue monoterpene concentrations and emission
rates in response to growth under increased [CO2 ] (Constable et al. 1999). This
lack of response was also observed in Adenostoma fasciculatum and Ceanothus
greggii (Baraldi et al. 2004) and Betula pendula (Vuorinen et al. 2005). Llorens
et al. (2009) investigated four monoterpene emitting “living fossil” gymnosperm and
one angiosperm tree species growing in a simulated Cretaceous polar environment
with doubled atmospheric CO2 concentration of 800 mol mol1 , and observed that
only the deciduous gymnosperm species were affected by high [CO2 ]. However,
there were interspecific differences with enhanced emissions in Metasequoia glyp-
tostroboides and inhibited emissions in Taxodium distichum, and variable responses
in Ginkgo biloba depending on the sampling date (Llorens et al. 2009). However,
it is not fully clear to which extend the monoterpene emissions in these deciduous
conifers came from storage or reflected de novo monoterpene synthesis.
The “immediate” emitter Quercus ilex exhibited an increase in monoterpene
emissions when grown under elevated [CO2 ] (Staudt et al. 2001). Results from the
study of Staudt et al. (2001) showed that leaves of Q. ilex in elevated [CO2 ] treatment
did not allocate more carbon to monoterpene production, but the emission capacity
of leaves increased together with the capacity of photosynthesis. In another studies
in Q. ilex, Loreto et al. (2001) found a slight inhibition of ’-pinene, “-pinene and
10 Modification of BVOC Emissions by Changes in Atmospheric [CO2 ]. . . 267

sabinene emission, but an increase of limonene emission. These differences were

correlated with changes in corresponding monoterpene synthase activities (Loreto
et al. 2001). Further studies are needed to gain insight into differential regulation of
different monoterpene synthases by elevated [CO2 ].

10.4 Effects of Elevated [O3 ] on Plant BVOC Emissions

BVOCs not only contribute to the formation of O3 and other oxidants (Sect. 10.1),
but O3 itself can influence the rate of BVOC emission from plants. There is a large
study-to-study variability in BVOC responses to [O3 ], and thus, no general response
direction of the effect of [O3 ] on isoprenoid emissions (Fig. 10.2). The evidence
has demonstrated that exposure of leaves to elevated [O3 ] can increase (Velikova
et al. 2005a, b; Fares et al. 2006) or decrease (Fares et al. 2006; Calfapietra et al.
2007, 2008) isoprene emission rates (Table 10.2). Here this controversial evidence
is analysed and possible explanations for the variability in the reported patterns
are given.

10.4.1 Influence of Acute Ozone Episodes

In laboratory experiments acute and short-term (300 nmol mol1 for 3 h) exposure
to O3 significantly increased isoprene emission by reed (Phragmites australis)
leaves (Velikova et al. 2008). Analogously, acute, high O3 concentrations have been
observed to stimulate monoterpene emissions from Quercus ilex leaves in laboratory
experiments, and this effect was apparent even for leaves that had developed in an
otherwise low [O3 ] atmosphere (Loreto et al. 2004).
In the case of stress-signalling volatiles and sesquiterpenes, fumigation with
[O3 ] also stimulated the emissions in Scots pine (Pinus sylvestris) and in tobacco
(Nicotiana tabacum) ozone-sensitive (Bel W3) and ozone–resistant (Bel B) cultivars
(Heiden et al. 1999). An ozone treatment of 120–170 nmol mol1 for 5 h induced
visible damage in the ozone-sensitive tobacco cultivar, while the more tolerant
cultivar was not affected (Heiden et al. 1999). Both cultivars emitted methyl
salicylate and a range of sesquiterpenes after the [O3 ] treatment, but the emission
of several C6 compounds of the lipoxygenase pathway (LOX, also called green leaf
volatiles; cis-3-hexen-1-ol as the most prominent one, trans-2-hexen-1-al, 1-hexanol
and cis-3-hexenyl acetate) was only observed in the ozone-sensitive cultivar (Heiden
et al. 1999). High O3 concentration of 120–200 nmol mol1 also induced emissions
of homo- and sesquiterpenes in lima bean (Phaseolus lunatus), a non-isoprene
emitter, when grown in the stirred tank reactors (Vuorinen et al. 2004). Following
exposures to different ozone concentrations from 80 to 1,700 nmol mol1 and
timing (1.0–8.8 h), typical LOX volatile products were emitted from ozone-sensitive
268 C. Calfapietra et al.

Table 10.2 Variability in BVOC emission responses to elevated ozone exposure in different
plant species and different fumigation experiments
Experimental
Species system Effect Reference
Isoprene emission
Short-term experiments
Phragmites australis Enclosed chamber C Velikova et al. (2005a, 2008)
Quercus pubescens LOF system , C, Da Velikova et al. (2005b)
Long-term experiments
Populus alba Enclosed chamber C,b Fares et al. (2006)
Populus tremuloides Aspen FACE , Dc Calfapietra et al. (2007, 2008)
Ginkgo biloba Open-top chamber C Li et al. (2009)
Monoterpene emission
Short-term experiments
Quercus ilex Enclosed chamber C Loreto et al. (2004)
Long-term experiments
Pinus sylvestris Continuously stirred C Heiden et al. (1999)
tank reactors
Ceratonia siliqua Open-top chamber C,, Dd Llusià et al. (2002)
Olea europaea,
Quercus ilex ssp. ilex
Quercus ilex ssp. ballota
Ginkgo biloba Open-top chamber C Li et al. (2009)
Other BVOC emission
Short-term experiments
Phaseolus lunatus Growth chamber Ce Vuorinen et al. (2004)
Nicotiana tabacum Continuously stirred Cf Beauchamp et al. (2005)
tank reactors
Long-term experiments
Nicotiana tabacum Continuously stirred Cg Heiden et al. (1999)
tank reactors
Pinus halepensis Open-top chamber C, Dh Peñuelas et al. (1999)
Solanum lycopersicum
Peatland microcosms Growth chamber Ci Rinnan et al. (2005)
The treatment time varied between 3 h and 10 days for short-term and from 30 days to 8 years
for long-term treatments
a
Dose effect, emission burst after recovery
b
Age-dependent effect
c
Decrease in the ozone-sensitive clone
d
Seasonality and species dependence
e
Homo- and sesquiterpenes
f
C6 -volatiles (LOX products)
g
Sesquiterpenes
h
Seasonal dependence
i
Non-methane BVOC

tobacco cultivar Bel W3 (Beauchamp et al. 2005). These LOX products are derived
from lipoxygenase activity after release of polyunsaturated free fatty acids from
membranes (Feussner and Wasternack 2002; Liavonchanka and Feussner 2006).
They are considered as products of membrane lipid peroxidation related to leaf
10 Modification of BVOC Emissions by Changes in Atmospheric [CO2 ]. . . 269

damage (Loreto et al. 2006). Thus, emissions of LOX products from plants exposed
to high [O3 ] during acute exposure episodes provide evidence that these acute
episodes result in physiological damage.

10.4.2 Localized [O3 ] Fumigation (LOF) System Experiments

Using a LOF system (Sect. 10.2.2), it was demonstrated that exposure to low
(60 nmol mol1 ) and intermediate (190 nmol mol1 ) O3 concentrations for three
consecutive days (9 h/day) did not affect isoprene emissions from pubescent
oak (Quercus pubescens) leaves (Velikova et al. 2005b). High O3 concentration
(300 nmol mol1 ) significantly reduced isoprene emission by leaves sampled imme-
diately after the treatment as compared with control leaves, but isoprene emission
rates were increased significantly in 288 h after termination of the high [O3 ]
treatment (Velikova et al. 2005b), suggesting that isoprene stimulation is delayed
relative to the onset of O3 stress, probably reflecting the activation of a whole class
of constitutive and induced genes (Sharkey et al. 2005). It is likely that the high
isoprene emissions help to quench reactive oxygen species (ROS) and to enhance the
membrane stability in leaves recovering from O3 stress (Possell and Loreto 2013).

10.4.3 Long-Term [O3 ] Fumigation Effects

Using open-top chamber experiments, Peñuelas et al. (1999) observed a significant

positive relationship between tropospheric O3 exposure and BVOC concentrations
in the atmosphere, which followed seasonal patterns with a maximum in early
summer. However, there were species-specific responses with no significant effect of
O3 on BVOC emissions in Aleppo pine (Pinus halepensis), but a highly significant
O3 effect in tomato (Solanum lycopersicum) (Peñuelas et al. 1999). In open
top chamber experiments with different Mediterranean woody plant species, an
overall enhancement of BVOC emissions under high O3 concentrations was shown,
indicating that a positive feedback on tropospheric O3 formation can be expected
(Llusiá et al. 2002). When considering specific BVOCs, there were variable results
among species and time of the year. Emission of ’-pinene decreased with [O3 ]
fumigation in Olea europaea, while ’-pinene and limonene emissions increased
in Quercus ilex (Llusiá et al. 2002).
Stimulation of isoprene emission after ozonation has been also observed in white
poplar (Populus alba) leaves (Fares et al. 2006). However, this increase of isoprene
emission was restricted to leaves that developed inside the cuvette during the ozone
treatment; when leaves that had developed outside the cuvette, in an atmosphere
with low [O3 ], were exposed to the higher treatment O3 concentration, isoprene
emission rate was reduced (Fares et al. 2006).
Long-term adaptation (4 months) to an O3 concentration of 80 nmol mol1
(9 h/day) increased the emissions of isoprene and monoterpenes from Ginkgo biloba
270 C. Calfapietra et al.

(Li et al. 2009). In Pinus sylvestris, exposure to elevated [O3 ] concentrations (daily
mean concentration of 50 nmol mol1 , 8 h/day) in open-top chambers for a period
of 2 years also led to a significant increase of monoterpene emissions, while no
visible damage to the needles was observed (Heiden et al. 1999). Analogously, in
a growth chamber experiment, high O3 concentration (150 nmol mol1 for 36 days
with 9 h/day) increased the emissions of all BVOCs that were emitted from a
peatland (Rinnan et al. 2005). Emissions of aromatics, terpenoids and N-containing
compounds were doubled at 150 nmol mol1 O3 levels (Rinnan et al. 2005).
In contrast to these observations, long-term field experiments showed a signifi-
cant decrease of isoprene emission in an O3 -sensitive aspen (Populus tremuloides)
clone, whereas smaller decreases in isoprene emissions were observed in the O3 -
tolerant clone (Calfapietra et al. 2008).

10.4.4 Mechanisms of Long-Term [O3 ] Influences

When considered across the entire range of experiments and species, these past
findings on the effect of short- and long-term [O3 ] exposures suggest that BVOC
emissions might be stimulated by acute O3 doses, although with species-dependent
and microenvironment-dependent variation. Chronic O3 exposure may inhibit
emissions, but the responses seem to be different for isoprene and monoterpenes
(Calfapietra et al. 2009, Fig. 10.2). The differences observed in acute and chronic
exposure experiments likely reflect important differences in the capacity for the pho-
tosynthetic systems of plants to tolerate oxidative stress, and also the biochemical
acclimation responses associated with alterations in gene expression. In the study
of Calfapietra et al. (2007), lower isoprene emission rate in the sensitive aspen
clone correlated with reduced isoprene synthase (IspS) mRNA level and reduced
concentrations of IspS protein in the leaves. Although the drop in IspS protein level
induced a drop in the isoprene emission rate under elevated [O3 ], other mechanisms
could also have contributed to the observed inhibition, such as the reduction in the
pool size of DMADP, the main substrate for the formation of isoprene (Calfapietra
et al. 2008). This may have occurred because of [O3 ]-induced damage to the pho-
tosynthetic apparatus, including the electron transport system and trans-thylakoid
proton gradient, both of which control the production of NADPH and ATP, which
are required for DMADP synthesis. Clearly, there is a need for more quantitative
studies looking into the biochemical limitations under different ozone exposures.

10.4.5 Mechanistic Approaches to Understand [O3 ] Response

Variation in ozone responses suggests that there is a need for experiments that
explicitly compare acute and chronic responses with the aim of identifying the
limits of ozone tolerance as driven by ozone dose, and short- and long-term emission
10 Modification of BVOC Emissions by Changes in Atmospheric [CO2 ]. . . 271

responses. In order to improve O3 dose/effect predictions, a mechanistic approach

has been developed, which is based on analyses that take into account the O3 flux
through stomata (Emberson et al. 2000). The leaf O3 uptake rate has been shown to
depend on species-specific or genotype-specific maximum and minimum stomatal
conductances (Fares et al. 2008). It also depends on environmental variables such
as light, temperature and water availability in the plant-soil system, all of which
modify leaf stomatal conductance (Löw et al. 2006). It has been demonstrated that
O3 uptake by strong isoprene emitter Populus nigra is limited by stomatal aperture,
and it was suggested that O3 removal due to reactions inside the leaves occurs much
faster than delivery of O3 through the stomata (Fares et al. 2008). Relatively low
reactivity of O3 with isoprene likely limits significant O3 losses in reactions in the
leaf-atmosphere boundary layer in isoprene-emitting species. On the other hand,
compared to isoprene, monoterpenes have greater potential to react with O3 within
or just above leaves (Atkinson 1997). The results obtained with Quercus ilex show
that gas-phase reactions may contribute substantially to O3 losses within the leaves
of monoterpene-emitting species (Fares et al. 2008). It was also shown that O3 flux
into the leaf may be driven by the reaction with monoterpenes (Loreto and Fares
2007) and other antioxidant molecules (Burkey and Eason 2002). In fact, O3 uptake
in the leaves of isoprenoid emitting species, such as Populus alba, Phragmites
australis and Quercus ilex, was higher than in the leaves of the non-emitting species
Nicotiana tabacum and Betula pendula (Loreto and Fares 2007). More studies are
needed to understand the biochemistry of O3 reactions inside the leaf to exploit
the capacity of vegetation to naturally scavenge O3 . At this point, it is interesting
that Jardine et al. (2012) showed significant emissions of oxygenated compounds
presumably coming from within-leaf gas-phase reactions between isoprene and
OH. It would be interesting to determine if similar emissions of reaction products
between O3 and monoterpenes come from the leaves of monoterpene-emitting
species such as Quercus ilex.

10.5 Inclusion of [CO2 ] and [O3 ] Effects in Models

Accurate prediction of BVOC changes to future air concentrations of CO2 and air
pollutants is necessary for estimating consequences and possible feedbacks to the
chemistry of the atmosphere (Guenther et al. 1995; Wang and Shallcross 2000; Karl
et al. 2004; Yokouchi and Ambe 2007; Lerdau 2007; Sitch et al. 2007). Improved
knowledge of the environmental and physiological factors that regulate BVOC
synthesis and emission has made it possible to develop different models that take
into account the response of isoprene emissions to changing CO2 concentrations
including both the direct effect on the emission rate and indirect effects on
photosynthesis and photosynthetic products required for isoprenoid biosynthesis
(Monson et al. 2012; Grote et al. 2013).
There is little information available on the combined effects of elevated CO2 and
elevated O3 concentrations on BVOC emission and the combination of these factors
272 C. Calfapietra et al.

with warming. These interactions are potentially relevant as there may be secondary
effects of [O3 ] and [CO2 ] on the temperature or light algorithms as opposed to direct
effects of these parameters on isoprene emission. For example, both elevated [CO2 ]
and [O3 ] can lead to reduced stomatal conductance. Low stomatal conductance can
increase leaf temperature by reducing latent heat loss. The [CO2 ]-dependent higher
leaf temperature (Zavala et al. 2013) might increase isoprene synthesis while [CO2 ]-
dependent reduced stomatal conductance allows isoprene to build up in the leaf
(Sharkey 1991; Fall and Monson 1992) thereby enhancing leaf thermotolerance (see
the chapter of Possell and Loreto 2013).

10.5.1 Simulating Atmospheric [CO2 ] Effects on Emissions

The effect of CO2 concentration on terpenoid emissions has been included in leaf-
level algorithms through relations to physiological processes and in particular to
photosynthetic and biochemical processes (Niinemets et al. 1999; Martin et al. 2000;
Zimmer et al. 2000; Bäck et al. 2005; Grote and Niinemets 2008; Wilkinson et al.
2009; Rasulov et al. 2009; Possell and Hewitt 2011). In most of the cases, increases
in CO2 concentration are associated with linear or non-linear decreases in isoprene
emissions (Possell et al. 2005; Wilkinson et al. 2009, Sect. 10.3). The shape of this
response can have profound influences on predictions of future isoprene emission in
elevated [CO2 ] atmospheres (Arneth et al. 2007; Heald et al. 2009). A few available
studies of CO2 effects on monoterpenes emissions show a more variable picture,
partly due to different responses of storage- and “non-storage”-emitters (Constable
et al. 1999; Staudt et al. 2001; Loreto et al. 2001; Snow et al. 2003; Baraldi et al.
2004; Vuorinen et al. 2005; Llorens et al. 2009).
Two factors that have been conflated in past models of isoprene emission
analysing its impact on air quality in future atmospheres of higher [CO2 ] are the
effects of CO2 on net primary production (NPP) (a positive effect, Norby et al.
2005), which can increase global emissions due to more emitting leaf biomass, and
the effect of [CO2 ] on isoprene emissions per unit of leaf area (a negative effect).
This conflation was noted by Monson et al. (2007), and as a result, many past
predictions of atmospheric chemistry in future atmospheres with elevated [CO2 ]
are highly uncertain. The contribution of elevated [CO2 ] to both NPP and isoprene
emissions must be considered in models aiming to project future trajectories of
atmospheric chemistry.
More recently, some global models have incorporated both the direct and the
indirect effects of elevated [CO2 ] on isoprenoid emissions using different models
and approaches. Arneth et al. (2008) have shown that in Europe we should expect
in most of the areas an increase in isoprene emission due to a stimulation of leaf
area index (LAI) and due to land-use change, mainly because of the changes in
the coverage of forested areas. They also showed that including the direct CO2
inhibitory effect will completely counteract the effect of a warmer climate, with
an overall decrease in the isoprene emission rate in most regions of Europe.
10 Modification of BVOC Emissions by Changes in Atmospheric [CO2 ]. . . 273

Heald et al. (2009) showed similar results at the global scale separating the
short-term effect of elevated [CO2 ] on isoprene emission and the long-term effect
of elevated [CO2 ] on plants and highlighting in particular that when changes in
vegetation distribution and leaf area density are included in models, future isoprene
emissions could increase by more than a factor of two.
It is clear that in assessing the sensitivity of isoprene emissions to future increases
in atmospheric CO2 concentration, we cannot ignore the parallel increases in
temperature that are likely to occur. The positive effect of global warming, in
terms of temperature and its effect on terrestrial productivity, tends to be of the
same magnitude and sometimes even higher than the CO2 inhibition of isoprene
production (Arneth et al. 2007, 2008; Heald et al. 2009; Young et al. 2009). For
instance large increases in the emission by the end of this century (27–70 % relative
to present-day emissions) are expected due to the effect of global warming and
more productive vegetation (Sanderson et al. 2003; Lathière et al. 2005; Wiedinmyer
et al. 2006; Arneth et al. 2008; Ashworth et al. 2013). Moreover the increase in the
frequency of extreme high temperature events will likely produce episodic peaks of
isoprene emission.
Several models have been built not only to predict future CO2 scenarios but
also to investigate past CO2 concentration effects on and temporal changes in
BVOC emissions. Recent studies suggest that isoprene emissions were generally
enhanced by low [CO2 ], but only at the leaf level, as the overall effect on
canopy isoprene emission was counterbalanced by reduced leaf area (Possell et
al. 2005). Simulations of BVOCs emissions since the Last Glacial Maximum
(LGM) showed that total emissions of isoprene and monoterpenes over Europe
increased due to higher temperatures and an increase of vegetation abundance;
despite the counteracting effect of CO2 inhibition, the relative increase in isoprene
and monoterpene emissions was larger than the increase in gross primary production
(GPP), due to the stronger temperature sensitivity of terpenoid production compared
to photosynthesis (Schurgers et al. 2009).
We note that it is important to couple the models predicting the effects of
increased CO2 concentrations on BVOC emissions with models predicting the
ground ozone levels, integrating the future scenarios of biogenic emissions with
air quality models and with regional climate data (Wiedinmyer et al. 2006; Young
et al. 2009; Pacifico et al. 2009).

10.5.2 Towards Models of [O3 ] Responses

Incorporation of the effects of air pollutants on isoprenoid emissions is more

complicated and requires new models that include a stress signalling component
(Niinemets et al. 2010c; Grote et al. 2013). Although such models are currently
under development (Grote et al. 2013), air pollutant effects cannot currently be
effectively simulated by process-based approaches. Nevertheless, at leaf level, an
hypothesis based on the short- and long-term studies of [O3 ] fumigation has been
274 C. Calfapietra et al.

formulated by Calfapietra et al. (2009) linking BVOC emission to the O3 dose

according to a hormetic-dose response characterized by a stimulation of BVOC
emission at low O3 doses and an inhibition at high O3 doses. Modelling the O3
effects over the long-term would result in an inhibition due to both decreased leaf
area and decreased emission at leaf level, which is exacerbated when it occurs
in combination with elevated [CO2 ] (Calfapietra et al. 2008). Nevertheless, more
experimental work is needed to test the generality of this hypothesis (Fig. 10.2).

10.5.3 Modelling Changes in Competitive Relations

as the Result of [O3 ] Elevation

Although simulation of [O3 ] responses is currently difficult, [O3 ] elevation is

involved in altering numerous biological interactions, including alteration of terres-
trial carbon sink (Ashworth et al. 2013) and multitrophic interactions (Holopainen
et al. 2013). Here we address the important feedback loop between ozone tolerance
and changes in community composition.
It is likely that rising [O3 ] will provide a competitive advantage for isoprene-
emitting species over non-emitting species because of the protection that isoprene
synthesis provides against O3 stress (Lerdau 2007). This could lead to a decrease
in forest biodiversity, with isoprene-emitting species becoming more abundant and
thus causing an increase of isoprene emissions at the global scale, with a strong pos-
itive feedback on tropospheric O3 concentration (Lerdau 2007; Laothawornkitkul
et al. 2009). On the other hand, higher O3 concentrations could shorten the growing
season and decrease standing leaf biomass (Karnosky et al. 2003). These secondary
effects could negatively affect isoprene emissions and counteract any positive
feedback generated by climate warming (Arneth et al. 2008). In summary, it is
evident that future efforts of modelers should include O3 effects and feedbacks with
BVOCs for a better understanding of BVOCs-O3 interactions in the future.

10.6 BVOCs-Air Pollution Interactions and Implications

in a Changing Atmosphere

The interactions between BVOCs and O3 pollution are complicated, especially if

we consider the long-term responses. On one hand, BVOC emissions contribute to
the formation of O3 and other photochemical pollutants through reactions involving
NOx and OH radicals (Fuentes et al. 1999). In addition to being a greenhouse
gas, O3 is also a toxic pollutant that significantly reduces crop and forest yields
worldwide and is responsible for human health problems during pollution episodes.
Ozone phytotoxicity may significantly accelerate future climate warming due to
reductions in the terrestrial carbon sink. On the other hand, BVOCs protect plants
10 Modification of BVOC Emissions by Changes in Atmospheric [CO2 ]. . . 275

Fig. 10.3 Effects of BVOC emissions (including mainly isoprene and monoterpenes) on the O3
formation and direct and indirect effects of increased CO2 and O3 concentrations on BVOC
emissions. The indirect effect of [CO2 ] and [O3 ] results from the influence of these greenhouse
gases on temperature. The figure demonstrates a positive loop resulting from the rise in temperature
and O3 on BVOC emissions. The overall positive effect is only partially alleviated by the O3 uptake
by plants, potentially negatively affecting emissions

against oxidative stress such as O3 (Vickers et al. 2009; Possell and Loreto 2013),
and it has been hypothesized that increasing levels of O3 might stimulate BVOC
synthesis and emission (Loreto et al. 2004). This aspect has raised the hypothesis of
a positive loop considering that increasing levels of O3 in the future will favor BVOC
emitter species because of the protective role of these compounds and this will result
in higher BVOC load into the atmosphere and higher O3 production (Lerdau 2007,
Sect. 10.5.3).
A positive dangerous loop could also occur in the future if there is a long-term O3
stimulation of BVOC emissions as has been consistently observed in the short term
(Fig. 10.3). Such a long-term enhancement as observed in some studies (Fig. 10.2)
is, however, not general. For example, measurements carried out in plants exposed
for several years to elevated [O3 ] at the AspenFACE site demonstrated inhibition of
isoprene emissions by [O3 ] (Calfapietra et al. 2008).
The interactions between BVOC emission and atmospheric composition are
complicated also by the recent findings related to the O3 uptake capacity by plants
276 C. Calfapietra et al.

(Fig. 10.3). Ozone uptake was higher in BVOC-emitting plants than in non-emitters
(Loreto and Fares 2007; Fares et al. 2010). However, what are the stomatal and
non-stomatal contributions of this uptake and how relevant this process is for
plant physiological activity and ambient ozone levels is still uncertain. It has been
confirmed, however, that reactions between BVOCs and reactive oxygen species
(ROS) occur already inside the leaves and that reaction products such as methyl
vinyl ketone and methacrolein are emitted by most plants (Jardine et al. 2012),
suggesting that a similar reactivity might also occur between BVOCs and O3 .
This topic is central to understanding the vegetation capacity for uptake of air
pollutants, particularly in urban environments (Nowak et al. 2006) where we might
currently significantly underestimate the abundance of BVOC emitters (Niinemets
and Peñuelas 2008; Owen et al. 2013). To conclusively assess the importance of
vegetation in oxidant uptake, an important task for future research is to determine
ozone uptake characteristics in relation to BVOC emissions for the main plant
species. This can be done by laboratory measurements controlling the physiological
status of the plant and the microclimatic conditions around it. Another key question,
considering all these interactions between BVOCs and O3 , is which effect is
stronger: the contribution of BVOCs to the O3 formation or the contribution of plants
(and even of BVOCs) on O3 removal. So far, the variabilities and uncertainties in
and understanding of the biological and chemical processes do not allow to draw
clear conclusions on the relevance of different processes and connecting feedback
loops.

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Chapter 11
Multitrophic Signalling in Polluted Atmospheres

Jarmo K. Holopainen, Anne-Marja Nerg, and James D. Blande

Abstract Volatile compounds emitted by plants in response to herbivory serve

as important cues within and between trophic levels, and as cues over more than
two trophic levels, such as in the attraction of enemies of herbivores. However,
many of the volatiles elicited by herbivory are highly reactive with key atmospheric
pollutants, implying that the signal is communicated over increasingly shorter
distances with increasing pollutant concentrations in the atmosphere. Thus, polluted
atmospheres can importantly alter the multitrophic interactions between trees, herbi-
vores and herbivore enemies. This chapter highlights the alterations in multitrophic
interactions and resulting modifications in plant fitness in polluted atmospheres.

11.1 Introduction

Atmospheric pollution is a major environmental factor and long known to have an

impact on plants (Bell and Treshow 2002), herbivores, especially insects (Docherty
et al. 1997), and the natural enemies of herbivores (Butler et al. 2009). Foresters
have seen these impacts in practice as trees languish and frequent insect outbreaks
occur in the surroundings of urban and industrial areas polluted by sulphur dioxide
(SO2 ) and oxides of nitrogen (NOX ). Heavy metal pollution distributed among
other air pollutants has often resulted in death of surrounding vegetation and
subsequent decline of communities of herbivores (Zvereva and Kozlov 2010) and
their natural enemies (Butler et al. 2009). Recent environmental and ecological
research has indicated that more ubiquitous air pollutants such as ozone (O3 ) will
affect vegetation over much longer distances from the urban and industrial point
sources (Sitch et al. 2007; Calfapietra et al. 2013 in this volume). This means that in

J.K. Holopainen () • A.-M. Nerg • J.D. Blande

Department of Environmental Science, University of Eastern Finland,
P.O. Box 1627, FI-70211 Kuopio, Finland
e-mail: [email protected]

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 285
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 11,
© Springer ScienceCBusiness Media Dordrecht 2013
286 J.K. Holopainen et al.

principle, all terrestrial ecosystems may suffer to a certain extent from air pollution
and subsequently air pollutants may globally disturb the biogenic volatile organic
compound (BVOC) signals evolved as facilitators of communication between plants
and plants and animals across multiple trophic levels.
This chapter is aimed at highlighting how polluted atmospheres can be involved
in altering multitrophic interactions communicated and mediated by BVOC signals
and how this may result in modifications in plant fitness. First we review the current
knowledge of the effects of air pollution on arthropod communities that live on
woody plants. Knowledge of multitrophic interactions in arboreal communities
under stress is needed for better understanding of BVOC signalling in forest
ecosystems and how much these signals are dependent on the health of trees.
The mechanisms of multitrophic signalling between plants, between plants and
herbivores and between plants and carnivores, which act as natural enemies of
herbivores, have been subjected to extensive research in recent years. However,
most of the studies have been conducted in controlled laboratory conditions and
less is known about how variability in air quality affects the volatile signals.
We will survey recent studies where the role of the atmospheric pollution load
is considered as an additional factor adding “noise” to BVOC signals. Then we
take a closer look at the impacts of different atmospheric pollutants, which have
distinctly different impacts on the lifetimes of the volatile signalling compounds in
the atmosphere. Three specific oxidants: ozone (O3 ) and OH and NO3 radicals react
with volatile signalling compounds produced by plants. These oxidants also occur in
less polluted environments, but at much lower concentrations than in areas currently
suffering from direct impacts of air pollution. Finally, we discuss future directions
for research on multitrophic signalling and how forecasted changes in atmospheric
conditions due to global change could impact the multitrophic signalling patterns. In
the coming decades, human population growth, increasing urbanization and global
climate change will extensively influence the environmental conditions for both
cultivated and wild plants. Therefore, a better understanding of the function and
pollutant sensitivity of BVOC-dependent multitrophic chemical signalling networks
is essential. In this chapter we primarily focus on stress-induced volatiles. For [O3 ]
effects on constitutive emissions, we refer to the Calfapietra et al. (2013) chapter in
this volume.

11.2 Impact of Air Pollution on Tree-Insect Interactions

Research on air pollution impacts on vegetation and interactions among species

in ecosystems has evolved in heavily industrialised areas where plants develop
visible pollutant stress symptoms. Trees as long-living plants tend to accumulate the
pollution load, especially species with long-living foliage. Accordingly, trees will be
more impacted by sustained pollution than annual plants. Several observations, the
oldest dating back to the year 1832 (Cramer 1951), of the surroundings of industrial
point sources have indicated that when forest trees are exposed to “smoke stress”,
11 Multitrophic Signalling in Polluted Atmospheres 287

containing sulphur dioxide (SO2 ) as the main gaseous air pollutant, outbreaks
of needle mining moths become more frequent (Oksanen et al. 1996; Kozlov
2003). Similar observations of increased densities of herbivorous insects on trees
in industrial environments have been made for sucking insects such as the bark-
feeding plant bug Aradus cinnamomeus (Heliövaara and Väisänen 1986) and aphids
(Villemant 1981; Holopainen et al. 1993). Higher aphid population densities and
increased growth rates of aphids have also been reported in trees along roads with
heavy traffic (Braun and Flückiger 1985; Bolsinger and Flückiger 1987). On the
other hand, investigations in industrial areas have indicated that defoliating insects
with chewing mouth parts do not usually have mass outbreaks on forest trees
exposed to high air pollutant loads (Heliövaara and Väisänen 1986; Holopainen and
Oksanen 1995). One explanation for the relatively low incidence of defoliators in
polluted atmospheres is that defoliators accumulate potentially harmful particulate
pollutants from the leaf surfaces in their bodies, while phloem feeders, cambium-
feeding bark beetles and mining species only respond to the quality changes of their
host plants under a pollutant load (Holopainen and Oksanen 1995).
Experimental exposures of plants to gaseous, semivolatile and precipitating
pollutants such as SO2 , NO2 , ammonium, nitric and sulphuric acid deposition
and fluorides have demonstrated a rapid increase in growth rate and number of
aphids on woody plants (e.g., Bolsinger and Flückiger 1989; Holopainen et al.
1991; Neuvonen and Lindgren 1987). This indicates that changes in plant quality,
such as a decrease in protein concentration and better availability of free amino
acids (Bolsinger and Flückiger 1989; Kainulainen et al. 1993) could be the major
reason for insect outbreaks on woody plants in areas with high air pollution. Foliar
secondary metabolites of trees including terpenoids such as monoterpenes and resin
acids (Kainulainen et al. 1993, 1994, 1995a, b) and phenolic compounds such as
total phenolics and tannins (Julkunen-Tiitto et al. 1995; Kainulainen et al. 1993,
1994, 1995a, b) have displayed less uniform responses to industrial air pollution
stress. The impact of the more ubiquitous pollutant ozone is known to induce the
production of simple phenolics and flavonoids (Lindroth 2010) and affect emissions
of volatile isoprenoids, especially in interaction with increases in atmospheric CO2
concentration (Peñuelas and Staudt 2010; Holopainen and Gershenzon 2010).
Besides the effects of air pollution on plant defence and nutritional quality for
herbivores, harmful impacts of pollutants on parasitoids (reviewed by Butler et al.
2009) and predators (Sorvari and Eeva 2010; Percy et al. 2002; Zvereva and Kozlov
2010) of herbivores may also result in rapid population growth of herbivores and
cause insect outbreaks. The impacts of pollutants on the third trophic level, and in
particular on insect parasitoids, have been variable depending on pollutant type and
species studied. A review of controlled-condition experiments has indicated that in
the majority of the studies, air pollutant load did not affect parasitoids (18 cases) or
had negative impacts (also 18 cases), and only in three cases, there were positive
effects of air pollutants on the performance of insect parasitoids (Butler et al.
2009). Most frequently, harmful effects on parasitoids were caused by ozone and
heavy metal pollution. In particular, extensive field-scale exposures of deciduous
forest trees to doubled ambient ozone levels over several years resulted in very
288 J.K. Holopainen et al.

high aphid populations relative to parasitoid densities (Percy et al. 2002). These
results suggest that high concentrations of oxidizing pollutants may have a negative
impact on tritrophic signalling mediated by herbivore-induced BVOCs in polluted
atmospheres.

11.3 Plant Volatiles as Signalling Compounds

Within and Across Trophic Levels

11.3.1 Volatile Compounds Induced by Biotic

and Mechanical Injury

The “scentscape” of BVOCs created by living plants and associated organisms plays
an important signalling function in species interactions and ecosystem processes
(McFrederick et al. 2009). When plant cells are damaged mechanically (Brilli
et al. 2011; Piesik et al. 2011) by herbivores (Blande et al. 2007; Allmann
and Baldwin 2010; Dicke and Baldwin 2010; Holopainen and Gershenzon 2010;
Schaub et al. 2010; Holopainen 2011; Piesik et al. 2011; Copolovici et al. 2011;
Clavijo McCormick et al. 2012; Trowbridge and Stoy 2013 in this volume),
by fungal pathogens (Vuorinen et al. 2007; Toome et al. 2010) or inoculated
with fungal symbionts (Schausberger et al. 2012), plants start to emit volatile
organic compounds (BVOCs) at higher rates. Mechanical damage of the foliage
of herbaceous plants and deciduous trees caused for example by chewing mouth
parts, typically results in the rapid emission of various green leaf volatiles (GLVs),
C6 volatile oxidation derivatives of fatty acids with a typical scent of cut grass
(Schaub et al. 2010; Clavijo McCormick et al. 2012; Monson 2013 in this
volume). Soon after these initial emissions, there are elevated emissions of other
compounds such as monoterpenes and sesquiterpenes (Schaub et al. 2010). Of the
herbivore-induced terpene compounds, acyclic homoterpenes, 4,8-dimethylnona-
1,3,7-triene (DMNT) and 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), are
among the most widespread volatiles produced by angiosperms (Tholl et al. 2011).
Homoterpenes can be found in flower emissions of many plant species (Tholl et al.
2011), but their greatest significance is in attracting parasitoids and predators of
herbivores to herbivore-damaged plants (Pinto et al. 2007a). In deciduous trees,
DMNT is the most responsive homoterpene in the BVOC bouquet of trees damaged
by chewing insect herbivores (Arimura et al. 2004; Blande et al. 2007, 2010b;
Staudt and Lhoutellier 2007; Mäntylä et al. 2008). Substantial methyl salicylate
(MeSA) emissions in deciduous trees, such as Betula pendula and Alnus glutinosa,
are indicative of aphid infestation (Blande et al. 2010b).
Conifers represent an interesting model system for studies of volatile emissions,
as they have a wide range of constitutive and induced compounds, as well as
a diversity of stored and constitutively emitted compounds. Among the elicited
compounds of conifers are those that are “novel”, mostly emitted only after damage
11 Multitrophic Signalling in Polluted Atmospheres 289

Fig. 11.1 Small spruce sawfly (Pristiphora abietina) is feeding on young developing needles of
Norway spruce (Picea abies) shoots. Herbivore-induced BVOC emission rates (pmol g1 DW s1 )
of young shoots have an altered profile with the most distinctive increase in the emission of
monoterpenes 3-carene, limonene, “-phellandrene and sesquiterpene (E)-“-farnesene (Blande et al.
unpublished data). Volatiles were collected from individual branches of Picea abies saplings that
had been either enclosed for five days in mesh bags containing 5–6 spruce sawfly larvae, or
enclosed in a mesh bag without larvae. Branches were enclosed in polyethylene terephthalate
(PET) bags and samples were collected onto Tenax TA for 30 min. All samples were analysed
by gas chromatography-mass spectrometry

of the plant by herbivores (e.g., Kännaste et al. 2008, 2009). The induced emissions
also include compounds that are normally emitted constitutively, but are found
in much higher quantities after herbivore damage. For example, when young
shoots of Norway spruce (Picea abies) are damaged by the little spruce sawfly
(Pristiphora abietina) there is a distinctive increase in the emissions of some
constitutively emitted monoterpenes such as limonene, 3-carene, “-pinene and ’-
pinene (Fig. 11.1). In addition, the monoterpenes terpinolene, linalool, borneol and
“-phellandrene and the aromatic compound methyl salicylate are “novel” inducible
compounds that are emitted only by branches damaged by sawfly larvae. The
sesquiterpene “-farnesene is highly inducible in P. abies, but these emissions may
also occur in minor quantities in intact control plants. In the shoot emissions of
Scots pine (Pinus sylvestris) seedlings suffering from pine weevil (Hylobius abietis)
induced stem bark injury, several monoterpenes and sesquiterpenes were emitted at
a significantly increased rate, but monoterpene 1,8-cineole emissions were absent in
290 J.K. Holopainen et al.

shoots of intact plants (Heijari et al. 2011). Significant increase of total monoterpene
emissions from herbivore damaged conifer foliage is mostly a result of mechanical
damage to needles and the resulting flow of resins from storage organs, and elevated
emissions continue until the wound sites are sealed by oxidized and polymerized
terpenes (Loreto et al. 2000). Mechanical damage simulating herbivory of needle
tissue results in a similar increase in total monoterpene emissions to that induced by
actual feeding by an insect herbivore (Litvak et al. 1999).

11.3.2 Volatile Compounds Involved in Within-Plant Signalling

A recently discovered primary signalling function of plant volatiles emitted by

branches of herbivore- or pathogen-attacked trees is to protect unwounded branches
from the risk of infestation by herbivores (Rodriguez-Saona et al. 2009) or infection
by pathogens (Yi et al. 2009). Such inter-branch signalling can result from the
full mixture of herbivore-induced BVOCs (Rodriguez-Saona et al. 2009) or from
specific herbivore-induced compounds such as methyl jasmonate, methyl salicylate
(Heil and Ton 2008) or cis-3-hexenyl acetate (Frost et al. 2008). Rodriguez-
Saona et al. (2009) showed that intact branches of highbush blueberry (Vaccinium
corymbosum) exposed to volatiles of gypsy moth (Lymantria dispar) damaged
branches had as much as 70 % less damage caused by larvae than branches exposed
to volatiles from undamaged branches and had greater chemical defences including
higher amounts of endogenous cis-jasmonic acid. One feature of this within-plant
signalling is that non-vascularly connected parts of the same plant may be primed
for enhanced defence responses if the given biotic stressor were to attack. Priming
undamaged parts of trees with a volatile signal is a more rapid and efficient method
of signalling than long-distance vascular signalling in trees (Frost et al. 2007). This
allows a systemic (whole plant) response to be achieved, as indicated in holm oak
(Quercus ilex) damaged by Lymantria dispar larvae (Staudt and Lhoutellier 2007).
Defence priming could be the primary function of inducible plant BVOCs for within
plant signalling.

11.3.3 Volatile Compounds in Intraspecific Plant

to Plant Signalling

In dense woody ecosystems, the branches of neighbouring trees of the same species
are positioned very close within tens of centimetres to a few metres and signals
from damaged trees have been shown to prime undamaged neighbours for enhanced
defence responses. The first reports of airborne between-plant signalling, in the
1980s (e.g., Baldwin and Schultz 1983), concerned trees and the phenomenon of
increased resistance in intact plants close to herbivore-damaged plants was referred
11 Multitrophic Signalling in Polluted Atmospheres 291

to as “talking trees” (Baldwin et al. 2006; Heil and Karban 2010). Since that early
discovery, between-plant volatile signalling has been detected in many agricultural
crop plants (Heil and Karban 2010). It has been suggested that between plant
signalling may be a result of ‘eavesdropping’ by the receiving tree due to the
emitting plant benefiting more by signalling to itself, or natural enemies of its pests,
rather than to a nearby competitor (Arimura et al. 2010; Karban 2011). Irrespective
of the fitness costs or benefits for the emitting plant, it is clear that the receiving
plants gain a benefit from being able to pre-empt herbivore or pathogen attack. The
genetic relatedness of the receiving plant to the emitter can importantly alter the
responsiveness of the receiver plant to the chemical signal. Recent studies have
indicated that sagebrush (Artemisia tridentata) plants produced clonally from a
common parent plant are better able to detect signals from a neighbour of the same
clone than from other, genetically more distant A. tridentata plants (Karban and
Shiojiri 2009; Ishizaki et al. 2012). This suggests that signalling between plants
via BVOCs could be far more sophisticated than previously thought with important
within population variations and evolutionary implications, and clearly will require
further experimental research.

11.3.4 Volatile Compounds in Interspecific Plant

to Plant Signalling

The closest association between two plant species is parasitism by parasitic plants, a
strategy represented by approximately 1 % of flowering plant species. Constitutively
emitted BVOCs of tomato (Solanum lycopersicum) are known to attract the parasitic
plant Cuscuta pentagona (Runyon et al. 2006). However, after infection, emissions
of constitutive and herbivore-induced BVOCs are attenuated so efficiently that
herbivore-damaged tomato plants become less attractive to parasitic plants (Runyon
et al. 2008). Parasitic plants can possibly use BVOCs as cues to avoid herbivore
damaged plants, which may have reduced quality as hosts. In addition, generalist
herbivores may damage the parasitic plant itself once they attack the infested
plant. Interspecific signalling, where volatiles that are released as the result of
mechanical damage or in response to herbivory affect the defence traits in another
plant species, “associative resistance”, has been reported for herbaceous plants
(Heil and Karban 2010). Himanen et al. (2010) reported that the woody shrub
Rhododendron tomentosum releases specific volatile monoterpenes and semivolatile
sesquiterpenes and sesquiterpene derivatives that can be adsorbed to the foliage
of neighbouring birch (Betula spp.) saplings and re-released into the atmosphere
when leaf temperatures are elevated in the morning. Furthermore, these semivolatile
compounds improved herbivore resistance in the receiver plant (Himanen et al.
2010). This raises the question of an opportunity cost of volatile release in the
emitter, i.e., whether the emitters reduce their own competitive status by having
healthier Betula spp. neighbours that are potentially more able to compete for shared
292 J.K. Holopainen et al.

resources? Or is this an indication of how strictly forest plant species depend upon
one another at the community level (Karban 2010)? Earlier, Choh et al. (2004)
reported that herbivore-induced semivolatile compounds can be adsorbed to the
surfaces of conspecific crop plants, and several studies have further demonstrated
certain uptake capacity of several monoterpenes (Copolovici et al. 2005; Noe et al.
2008). However, we do not yet know if these volatiles and semivolatiles adsorbed
onto foliage surfaces and/or solubilised in the lipid phase inside the leaves induce
defence responses in receiver plants or if they are just increasing defence by passive
evaporation and serve as repellents of herbivores or as attractants of herbivore
enemies.

11.3.5 Plant Volatile Compounds in Coevolution of Plants

and Herbivores – Direct Plant Defences

The coevolutionary arms race, antagonistic adaptations and counter-adaptations

between plants and their parasites, pathogens and herbivores, is a traditional way to
view plant–enemy interactions and evolution of defence traits in plants. However,
this coevolution is only expected when a plant species interacts with a specialist
herbivore and with specialist parasites such as fungal pathogens, which have enough
continuous pressure to reduce the fitness of the host plant (Agrawal and Heil 2012).
For example, a new secondary metabolite produced by a plant should reduce the
fitness of a specialist herbivore until a new detoxification trait has evolved in
the herbivore species. This detoxification trait is a competitive advantage for the
herbivore and it might consequently face less competition from other herbivore
species. This will lead to further tightening of the specialization of the herbivore
to the specific host plant species. Typically, specialist herbivores have broken the
defence barriers of a plant species, while the arms race may increase the plant
defence against generalist herbivores (Agrawal and Heil 2012). Generalists, on the
other hand, often specialize at feeding on specific plant organs (Schoonhoven et al.
2005) such as fast growing shoots, which may be less defended due to dilution of
defence compounds such as phenolic compounds in fast growing tissues (Keski-
Saari and Julkunen-Tiitto 2003).
Other plant resistance traits against insect herbivores include the emission of
feeding inhibitory or repellent BVOCs, which have impacts on herbivore preference
and host selection (Kloth et al. 2012; Clavijo McCormick et al. 2012). The same
specific volatile compounds that have primary attractant properties for specialist
herbivores act as repellents for other herbivores (Reddy and Guerrero 2004).
Species-specific plant BVOCs are most effective against specialist herbivores that
specifically target other plant families not constitutively releasing these compounds
(Himanen et al. 2010). Many of these species-specific constitutively emitted
volatiles are common for one plant taxonomic group, but appear less frequently or
not at all in other plant taxonomic groups. For example, the sesquiterpene alcohol
palustrol is found mainly only in Rhododendron spp. (Jaenson et al. 2005) and
11 Multitrophic Signalling in Polluted Atmospheres 293

volatile hydrolysis products of glucosinolates in the Brassicaceae (Tansey et al.

2010). Thus, if these compounds are emitted from species not synthesising them,
this can significantly reduce the host plant recognition by specialist herbivores.
In addition, species-specific plant BVOCs are in many cases effective against
generalist herbivore species as well and may even protect neighbouring plant species
from generalist herbivores in plant communities (Himanen et al. 2010). More
complicated repellent functions have been found for some specific volatiles, such
as 4-allyl anisole, a common compound in conifer species, which interrupts bark
beetle responses to their own pheromones (Reddy and Guerrero 2004).

11.3.6 Plant Volatile Compounds in Coevolution of Plants

and Carnivores – Indirect Plant Defences

Multitrophic signalling by plants is most often defined in a tritrophic context (Vet

and Dicke 1992), whereby parasitoids and predators of herbivores use herbivore-
induced BVOCs as orientation cues to find suitable herbivore hosts. Even the
hyperparasitoids of the fourth trophic level (parasitoids of a herbivore’s parasitoids)
can utilise herbivore-induced plant volatiles (Buitenhuis et al. 2005). It has also been
demonstrated, in a brassicaceous system, that parasitism of feeding herbivores can
have a significant effect on plants’ susceptibility to oviposition by latterly foraging
herbivores (Poelman et al. 2011a). In fact, the identity of the parasitoid species has
a greater effect on plant susceptibility than the identity of the herbivore (Poelman
et al. 2011a), which may be facilitated by the blend of volatile chemicals induced
by the differently parasitized herbivores, or through alternative changes to the plant
phenotype (Poelman et al. 2011b).
Most of the detailed research on tritrophic BVOC-mediated communication
between plants and natural enemies of herbivores has been conducted with herba-
ceous plants. There is still ample evidence that attraction of parasitoids (Wei et al.
2008) or predators (Scutareanu et al. 1997) of herbivores by plant BVOCs does
occur in woody plants under natural conditions, and is an effective strategy to
reduce herbivore damage (Ammunet et al. 2009; Babin-Fenske and Anand 2011;
Klemola et al. 2012). However, there has been some criticism that parasitoid larvae
inside a herbivore larva may stimulate feeding and increase the size of leaf area
consumed by the herbivore (Coleman et al. 1999). It has previously been shown that
parasitism of a galling psyllid (Baccharopelma dracunculifoliae) by a koinobiont
parasitoid (Psyllaephagus baccharidis) can stimulate nymph feeding and result
in larger galls, which are stronger nutrient sinks for plants (Espı́rito-Santo et al.
2004). It has also been shown that parasitism by a tachinid (Thelaira americana)
induces its host caterpillar (Platyprepia virginalis) to shift host from lupine (Lupinus
arboreus) to hemlock (Conium maculatum), thus increasing survival rates of the
caterpillar and reducing immediate feeding damage to lupine (Karban and English-
Loeb 1997). However, parasitism of the frequent defoliator of mountain birch
294 J.K. Holopainen et al.

(Betula pubescens ssp. czerepanovii), the autumnal moth (Epirrita autumnata), by

the solitary endoparasitoid Zele deceptor, reduced leaf consumption significantly
and also hastened the onset of pupation in autumnal moth larvae (Ammunet et al.
2009). The question of whether parasitism of a plant’s herbivores ultimately benefits
the plant is complex, and only likely to be conclusively answered on a case by case
basis. Clearly, those parasitoids that reduce feeding damage by herbivores provide a
tangible benefit to the plant. Those parasitoids that promote feeding damage might
provide a long-term benefit by removing the oviposition potential of the herbivore.
However, if the plant is a short-lived annual such a benefit might not occur. More
research is needed to address the long-term health costs or benefits in plants that
support herbivores that are parasitized by feeding-promoting parasitoids.

11.4 Polluted Atmosphere Effects on Plant Volatile Signals

11.4.1 Reactivity of Plant Volatiles

In natural environments, many of the oxidative air pollutants appear in lower

concentrations than in industrial, urban and in heavy traffic polluted areas, but
the reactivity of biogenic BVOCs with air pollutants is probably an integral part
of the evolved functions of BVOCs (Lerdau and Slobodkin 2002), giving spatial
and temporal dimensions to the signals they convey. The atmospheric gas-phase
degradation of BVOCs produced by biogenic or anthropogenic sources is initiated
by reaction with hydroxyl (OH) radicals, ozone (O3 ) and nitrate (NO3 ) radicals or
via photolysis (Hallquist et al. 2009). Ozone reacts only by addition to C-C double
bonds of alkenes followed by degradation of the resulting ozonide (Pinto et al.
2010; Atkinson and Arey 2003). Many of the biogenic BVOCs emitted by plants,
such as monoterpenes and sesquiterpenes, contain one or more double bonds, which
explain their reactivity. OH and NO3 radicals also cleave C-C double bonds, but they
additionally affect C-H bonds and to a much lesser extent O–H bonds (Atkinson and
Arey 2003). The reactivity of different plant volatiles differs more than 3 (reactions
with ozone) to 5–6 (reactions with OH and NO3 radicals) orders of magnitude
(Table 11.1), implying that air pollution can importantly alter the signal strength,
composition and dispersal.

11.4.2 Loss of Volatile Signals Versus Possible New Signals

Processes related to degradation of biogenic BVOCs by ozone have been most

thoroughly studied (see Pinto et al. 2010 for a review). The concentrations of many
herbivore-induced compounds were decreased in ozone-rich atmospheres much
faster than in filtered air (Pinto et al. 2007a, b, c). Some of the reaction products
11 Multitrophic Signalling in Polluted Atmospheres 295

Table 11.1 Atmospheric lifetimes of selected constitutively emitted and herbivore-induced

biogenic volatile organic compounds in reactions with major reactive air pollutants
BVOC Lifetimes for reaction with oxidants
Compound Class OHa O3 b NO3 c Reference
Typical constitutively emitted
compounds
Isoprene I 1.4 h 1.3 day 1.6 h (1)
3-Carene MT 1.6 h 11 h 7 min (1)
Limonene MT 49 min 2.0 h 5 min (1)
’-Pinene MT 2.6 h 4.6 h 11 min (1)
Myrcene MT 39 min 50 min 6 min (1)
Longifolene SQT 2.9 h >33 day 1.6 h (1)
Methanol Alcohol 12 day >4.5 year 2.0 year (1)
Typical herbivory-induced
compounds
cis-/trans-Ocimene MT 33 min 44 min 3 min (1)
“-Phellandrene MT 50 min 8.4 h 8 min (1)
Linalool MT 52 min 55 min 6 min (1)
“-Caryophyllene SQT 42 min 2 min 3 min (1)
“-Farnesene SQT 52 min 26 min – (2)
DMNT (4,8-dimethyl-1,3,7 HT 40 min 60 min 3 min (3)
nonatriene)
TMTT (4,8,12-trimethyl- HT 30 min 30 min 2 min (3)
1,3,7,11-tridecatetraene)
cis-3-Hexenyl acetate GLV 1.8 h 7.3 h 4.5 h (1)
cis-3-Hexen-1-ol GLV 1.3 h 6.2 h 4.1 h (1)
cis-3-Hexenal GLV 11.2 day 3.0 h – (2)
Methyl salicylate Aromatics 73.5 h >9.8 year – (2)
References: (1) Atkinson and Arey (2003), (2) Arneth and Niinemets (2010), (3) Roger
Atkinson, personal communication
BVOC classes: I – isoprene, MT – monoterpene, SQT – sesquiterpene, HT – homoterpene,
GLV – green leaf volatile
Pollutant concentrations used in calculations:
a
Assumed OH radical concentration: 2.0  106 molecule cm3 (0.074 pmol mol1 ), 12-h
daytime average
b
Assumed O3 concentration: 7  1011 molecule cm3 (26 nmol mol1 ), 24-h average
c
Assumed NO3 radical concentration: 2.5  108 molecule cm3 (9.3 pmol mol1 ), 12-h night-
time average

of the initial oxidation step of BVOCs contain one or more polar oxygenated
functional groups, such as aldehyde, ketone, hydroxyl or carboxy groups. Oxidation
tends to make the products less volatile and more water soluble (Atkinson and
Arey 2003; Kroll and Seinfeld 2008; Harley 2013 in this volume). These reaction
products may have the potential to function as herbivore repellents when aged and
accumulated on plant surfaces (Signoretti et al. 2012a). For example, measure-
ments of the early reaction products of ’-pinene show that several organic acids,
including 10-hydroxypinonic acid, 10-hydroxypinonaldehyde, 4-oxopinonic acid,
296 J.K. Holopainen et al.

Fig. 11.2 Schematic representation of atmospheric behaviour of monoterpenes (MTs) and

sesquiterpenes (SQTs) and their possible impact on multitrophic interactions in clean and polluted
environments. The left side of the figure illustrates the situation in clean air where most of the
experimental behavioural studies have been conducted. These studies have shown that herbivore
damage induces emissions of MTs and SQTs from plants, and natural enemies of herbivores
use these compounds as orientation cues. The right side illustrates the situation in polluted
atmospheres. Formation of secondary aerosol particles from reactions of MTs and SQTs with O3 ,
OH and NO3 radicals have been shown to be much higher in elevated oxidant concentrations
than in normal background concentrations in reaction chamber studies. There is some evidence
that degradation of herbivore-induced BVOCs in polluted air disturbs the orientation behaviour
of parasitoids and BVOC-transmitted defence priming in neighbouring plants. So far unexplored
area of research is how reaction products of BVOCs, such as oxidized semivolatiles condensed on
leaf surfaces and secondary organic aerosol (SOA) particles deposited onto vegetation, may affect
herbivore behaviour

and 10-oxopinonic acid are observed in the early stages of atmospheric reactions
(Jaoui and Kamens 2001). These organic acids may play an important role in the
early phase of secondary aerosol formation. Further oxidation may also lead to
formation of solid aerosol particles (Fig. 11.2; Cape 2008; Kroll and Seinfeld 2008;
Virtanen et al. 2010) or fragmentation to lower molecular weight volatiles, which
finally oxidize to CO2 (Atkinson and Arey 2003).
The complexity of atmospheric reactions of BVOCs with oxidants is best
illustrated by the reaction chamber study by Kundu et al. (2012). They analysed the
organic reaction products of the monoterpene limonene mixed with O3 in a reaction
chamber and found approximately 1,200 different organic compounds formed from
11 Multitrophic Signalling in Polluted Atmospheres 297

this single BVOC. Due to the ubiquitous presence of O3 , and OH and NO3 radicals
in the atmosphere, it is highly probable that some of the reaction products of
oxidation of limonene or other major BVOCs emitted by herbivore-damaged plants
may have similar biological functionality to the original compounds synthesised by
plants. If the olfactory sensors of predators and parasitoids are able to distinguish
between the original compounds released by a damaged plant and the oxidant
reaction products of the originally emitted compounds as suggested by Pinto et al.
(2007a), the ratio of these compounds could be indicative of the distance from the
BVOC source and the location of a potential host herbivore. Electro-antennogram
(EAG) recordings are needed to elucidate if the ratios of the original herbivore-
induced compounds and their reaction products can act as functional signals for
insects.
The nitrate (NO3 ) radical is formed in reactions of O3 with NOX (NO and NO2 )
that can be of soil, plant or anthropogenic origin. NO3 radicals undergo very rapid
photolysis (5 s) in direct sunlight (Atkinson and Arey 2003). Therefore, NO3
concentrations are usually only at measurable levels during the nighttime (Atkinson
and Arey 2003). NO3 concentrations are found at the highest levels within tree
canopies, where nighttime concentrations of NO3 can reach 20 ppt (McFrederick
et al. 2009). This is very important for the signalling capacity of many herbivore-
induced BVOCs, since many of these compounds are more reactive with NO3
radicals than with OH radicals or ozone (Table 11.1, Fig. 11.2). Damaged plants are
able to compensate for the high reactivity of some herbivore-induced compounds,
such as DMNT, with higher emissions of the compound during nocturnal herbivore
feeding compared to daytime feeding (Signoretti et al. 2012b). On the other hand,
emissions of herbivore-induced monoterpenes, such as “-ocimene (Copolovici et al.
2011), can be strongly light-dependent (Owen et al. 2002), and accordingly, the
way and extent to which atmospheric pollutants alter plant signals can be strongly
species-dependent.

11.4.3 Alterations in Signalling Distance

McFrederick et al. (2008) modelled the lifetimes of volatiles released from flowering
plants and estimated that the signalling distance of highly reactive floral BVOCs,
utilised by pollinators and constituting signals from first to second trophic levels,
may have decreased from kilometres during pre-industrial times to 200 m in the
more polluted conditions of present times. In their BVOC models, they used the
volatile monoterpenes “-ocimene, “-myrcene and linalool, which are common in
floral emissions, but increased emissions of the same compounds can be induced
by herbivore feeding, e.g., in black alder (Alnus glutinosa) (Copolovici et al. 2011),
hybrid aspen (Populus tremula x tremuloides) (Blande et al. 2007), hybrid poplar
(Populus x euramericana) (Brilli et al. 2009), holm oak (Quercus ilex) (Staudt and
Lhoutellier 2007), silver birch (Betula pendula) (Vuorinen et al. 2007), mountain
birch (Betula pubescens ssp. czerepanovii) (Mäntylä et al. 2008) or Scots pine
298 J.K. Holopainen et al.

(Pinus sylvestris) (Heijari et al. 2011). Further modelling (McFrederick et al. 2009)
showed that with increasing concentrations of ozone, the destruction rate of the
sesquiterpene “-caryophyllene was much faster than the destruction rate of the
monoterpenes “-ocimene and “-pinene, but with elevated concentrations of OH
and NO3 radicals the destruction rate of “-ocimene was similar to that of “-
caryophyllene (Table 11.1, Fig. 11.2). Although the research community tends
to think that all sesquiterpenes are very reactive and difficult to detect in plant
emissions, some sesquiterpenes such as longifolene have actually relatively long
lifetimes in polluted atmospheres (Arneth and Niinemets 2010).

11.4.4 Implications for Signalling Experiments in Polluted

Atmospheres

The possibility, that BVOCs exhibiting low reactivity can act as signals when more
highly reactive BVOCs have been oxidized, highlights the importance of includ-
ing other naturally occurring oxidants such as OH radicals in such experiments
(McFrederick et al. 2009). Pinto et al. (2007a) hypothesized that methyl salicylate
may provide a robust long distance signal, as it is relatively non-reactive with O3
and OH radicals (Canosa-Mas et al. 2002; Arneth and Niinemets 2010, Table 11.1).
So far, the experimental data for reactivity of methyl salicylate with NO3 radicals
are missing. However, assuming that the rate constant of methyl salicylate for the
reaction with NO3 radicals is similar to that for phenol and NO3 pair, methyl salicy-
late atmospheric lifetime is predicted to be approximately only 6 min for nighttime
oxidation initiated by NO3 radicals (Canosa-Mas et al. 2002). It may be possible to
create and control OH radicals in chamber and olfactometry experiments and thus,
simulate pollution-induced degradation of BVOCs. In chamber experiments, this
can be done by adding O3 and OH radical scavengers such as tetramethylethylene
(TME), 2-butanol or trans-2-butene to control OH and O3 levels (Arnts 2008; Hao
et al. 2011). Behavioural experiments with carnivorous arthropods will be more
complicated, because these compounds may directly affect the behaviour of insects
and mites, which is something that should be tested first.

11.5 Influence of Air Pollution on Multitrophic

Communication

11.5.1 Oxidative Pollutant Effects

As reviewed in Sect. 11.3, there is a multitude of within- and among-species

ecological interactions that rely on signalling via plant-emitted BVOCs. Earlier field
studies have clearly documented disturbances in many of these type of interactions
in forest ecosystems suffering from air pollution (Percy et al. 2002; Butler et al.
11 Multitrophic Signalling in Polluted Atmospheres 299

2009). Surprisingly few studies have tried to assess the mechanisms underlying the
disturbance and clarify the possible role of degradation of BVOC signalling in these
interactions (Holopainen and Gershenzon 2010). It is known that exposing plants
to higher concentrations of ozone can induce the emission of volatiles that are very
similar to those induced by spider mite feeding (Vuorinen et al. 2004a). However,
predatory mites are still able to distinguish between ozone and spider mite–induced
BVOCs when behavioural tests are run in purified air conditions, whereby there is no
loss of volatile signals due to degradation by residual ozone in the system (Vuorinen
et al. 2004a, b). Exposure of woody plants to elevated ozone concentrations also
induces BVOC emissions (Heiden et al. 1999; Loreto et al. 2004), but the impact of
these emissions on the behaviour of herbivores or their natural enemies has not been
assessed.
Studies using herbaceous plants and decaying plant material have indicated
that air pollutants may affect BVOC-based multitrophic communication between
host plant, herbivores and parasitoids or predators. Gate et al. (1995) showed
in chamber experiments using a fruit fly (Drosophila subobscura), which feeds
on decaying plant and fungal material, that searching efficiency of the braconid
parasitoid (Asobara tabida) was significantly reduced in ozone-rich air, resulting
in an approximately 10 % lower parasitism rate of D. subobscura. In chambers,
exposure to elevated SO2 and NO2 concentrations did not affect parasitoid behaviour
or parasitism rate of the host fly compared to clean air conditions (Gate et al.
1995). The authors considered this to be clear evidence that increased O3 levels
may interfere with the olfactory responses of the parasitoids.
There are few empirical studies that have assessed the effects of air pollution
on the measured levels of BVOCs and the associated behavioural responses of
animals of different trophic levels. So far, most of the existing studies have also
been conducted with non-woody plants. There is recent evidence from a modelling
study of forest trees in Canada that the normal outbreak cycle of the forest tent
caterpillar (Malacosoma disstria), which lasts approximately 10 years has been
disturbed, and in some air-pollution-stressed regions, there has been more severe and
frequent defoliation (Babin-Fenske and Anand 2011). Reduced parasitoid efficiency
related to chemical cue interference was found to be the most effective parameter
explaining the more frequent M. disstria outbreaks (Babin-Fenske and Anand 2011).
This finding suggests that the role of plant volatiles induced by key tree defoliators
in tritrophic communication should be investigated experimentally using the major
parasitoids and predators of the defoliators in polluted conditions.
A series of studies with lima bean (Phaseolus lunatus) and brassicaceous plants
has shown that high ozone concentrations can change the composition of herbivore-
induced plant volatiles (Pinto et al. 2007a, b, c, 2008; Himanen et al. 2009) and
may alter tritrophic interactions by influencing the behaviour of the natural enemies
of herbivores when behavioural tests are run in elevated ozone atmospheres (Pinto
et al. 2007c; Himanen et al. 2009). Y-tube assays were used to demonstrate that
parasitoids could still use herbivore-induced plant volatiles to locate hosts in the
presence of O3 , but given a choice between intact signals and O3 -degraded signals,
they preferred the intact signal (Pinto et al. 2007a, c). A field orientation test
300 J.K. Holopainen et al.

with the parasitoid wasp Cotesia vestalis, a major parasitoid of the diamond back
moth (Plutella xylostella), in an open-field ozone-exposure facility with ozone
concentrations enriched to double the ambient levels (Pinto et al. 2008) did not
indicate significant differences between ambient and O3 -enriched environments
either in the number of wasps found in the field, or in the percentages of parasitized
larvae. This result suggests that moderately elevated O3 will not affect the behaviour
of this parasitoid over a scale of a few metres, but does not exclude the potential
impact of ozone on orientation over longer distances, where the BVOC may be
degraded to a greater extent (McFrederick et al. 2009).
McFrederick et al. (2009) suggested that the laboratory results, showing a
significant reduction of signal compounds, but still not disturbing the behavioural
response of carnivores at the third trophic level, highlight two possible and not
mutually exclusive mechanisms that are responsible for the observed O3 effects.
Their first suggestion was that the products of the oxidation of the emitted chemicals
are not as effective as signals as the original scents, but can still provide some
signal. The second possibility is that BVOCs that exhibit low reactivity can act as
signals when more highly reactive BVOCs have been oxidized, and therefore are
important compounds for longer distance signalling. Recent chamber experiments
to measure plant to plant signalling in ozone rich air (Blande et al. 2010a) suggest
a third possible explanation. Blande et al. (2010a) found that receiver plants are
able to detect damaged neighbouring plants when grown in low ozone atmospheres.
When grown at 80 ppb ozone concentrations, the response of the intact receiver
plants at a 70 cm distance from the emitter plants was lost. This was most likely
due to several herbivore-induced signalling BVOCs being degraded in the airspace
between the plants. Plants do not appear to be very sensitive receivers of BVOC
signals and need higher concentrations of volatiles to elicit responses than insects
and mites. This suggests that the Y-tube result by Pinto et al. (2007a), showing very
low or undetected concentrations of some herbivore-induced BVOCs entering the
Y-tube, but still exhibiting behavioural responses by the third trophic level, may
indicate that insects and mites are able to detect herbivore-induced plant volatiles
in concentrations that are below the detection threshold of the GC-MS method
employed.

11.5.2 SO2 and Sulphur Depositions

Sulphur dioxide (SO2 ) is a primary pollutant related to industrial activity. SO2

concentrations peaked in Europe and North America in the late 1970s (Cape et al.
2003). Due to various clean air policies, the acute and chronic effects of SO2 on
vegetation, which were observed close to the emission sources in industrialised
countries and resulted in acid rains in remote areas relatively far from the emission
sources, have largely disappeared. However, rapid industrialisation in other parts
of the world is leading to localized and regional pollution hotspots with SO2
concentrations well in excess of thresholds for vegetation damage (Cape et al. 2003).
11 Multitrophic Signalling in Polluted Atmospheres 301

China has been the world’s largest emitter of SO2 since 2005 (Su et al. 2011).
Observations, both anecdotal and scientific, of increased herbivore presence on trees
close to pollution sources have indicated an effect of such pollutants, including SO2 ,
on plant-herbivore interactions. This is particularly true for herbivores that do not
feed on the surface tissues of leaves, but rather have strategies such as leaf mining
(Oksanen et al. 1996; Kozlov 2003) or phloem feeding (Holopainen et al. 1991;
Bell et al. 2011), although some studies indicate increased performance of chewing
herbivores at polluted sites (Bell et al. 2011). As sulphur deposition is an important
nutrient for plants, these observations are likely due to changes in the nutritional
quality of plants. For example, in needles of Scots pine (Pinus sylvestris), increased
concentrations of sucrose and reduced concentrations of glucose and fructose have
been observed (Kainulainen et al. 1995a), reflecting impaired sink-source relations
in polluted plants.
Direct impacts of SO2 on plant secondary metabolites have been studied to some
extent. Concentration of some monoterpenes and diterpenes in Pinus sylvestris
needles were reduced close to an industrial SO2 point source (Kainulainen et al.
1993). In contrast, Judzentiene et al. (2007) found that diterpene contents of
young needles of P. sylvestris increased with distance from a point source of
SO2 . Exposure of P. sylvestris and Picea abies seedlings to various concentrations
of SO2 , NO2 or ozone did not affect monoterpene concentrations in needles
(Kainulainen et al. 1995b). However, increased concentrations of the monoterpene
’-phellandrene and hydroperoxy conjugated dienes were found in the leaves of
the peppercorn tree (Schinus areira) when grown in an environment with high
SO2 concentration (Wannaz et al. 2003). To our knowledge, modified emissions
of isoprenoids or other BVOCs from plants exposed to elevated SO2 concentrations
have not been reported.
The positive effect of SO2 exposure on herbivore performance does not appear
to greatly affect the performance of parasitoids, with most studies so far indicating
mixed effects or no effect of SO2 on parasitoid performance (Butler et al. 2009).
However, in polluted atmospheres, SO2 is eventually oxidized to form sulphuric
acid, which in the liquid phase promotes oxidation of isoprenoids and leads to
formation of secondary organic aerosols (Hallquist et al. 2009). Sulphates formed
from SO2 may also act as condensation seeds for aerosol particles (Kroll and
Seinfeld 2008; Cape 2008) and increase condensation of most volatile organic
compounds (Kroll and Seinfeld 2008). These effects may significantly decrease the
atmospheric lifetime of herbivore-induced volatiles and thus affect the longevity of
the “cry for help” signals of herbivore-attacked trees (Holopainen 2011).

11.5.3 Interactions with Heavy Metal Pollution

Heavy metal pollutants are spread in atmospheric emissions over industrial areas
and they are deposited mainly on the soil surface where they are taken up by
plant roots. Heavy metals are also deposited on the surfaces of plant foliage. The
302 J.K. Holopainen et al.

direct phytotoxic effects of heavy metal pollution on plants are generally visible
as reduced photosynthesis, stunted growth and formation of growth abnormalities,
which collectively weaken the plants and their defence mechanisms (Aydin et al.
2012). However, Winter et al. (2012) reported that hydroponically grown maize
(Zea mays) exposed to either a high or low concentration of either Cu or Cd
displayed these visible symptoms, but the higher Cu dose was found to prime for
enhanced volatile production that can be triggered by caterpillar feeding. Cu stress
correlated with increased levels of reactive oxygen species in roots and priming
of herbivore-induced jasmonic acid in leaves. This result suggests that priming in
plants exposed to air pollutants in industrial environments rich with heavy metal
depositions can partly compensate for the loss of multitrophic BVOC signals in
polluted atmospheres by more intense defence response and higher emission rates
of herbivore-induced BVOCs.

11.5.4 Multitrophic Interactions Under Elevated

CO2 Atmospheres

Rising atmospheric CO2 concentration, [CO2 ], is affecting ecosystems globally,

but impacts of elevated [CO2 ] on multitrophic communication have rarely been
investigated. There is general knowledge that plant BVOC emissions are in many
cases lower in plants grown at elevated [CO2 ] levels than those grown at ambient
CO2 concentrations (Peñuelas and Staudt 2010; Fineschi et al. 2013; Calfapietra
et al. 2013 in this volume). However, evidence from CO2 exposure experiments with
parasitoids of Lymantria dispar suggests that the effects of elevated [CO2 ] on the
third trophic level are minor (Roth and Lindroth 1995). Experiments with deciduous
trees in open-field exposure systems with elevated CO2 and O3 concentrations
(Percy et al. 2002) indicated reduced population sizes of natural enemies of aphids
in elevated [O3 ] plots, and no effects of elevated [CO2 ] supporting, the observations
of Roth and Lindroth (1995).
In small-scale olfactometry experiments, Vuorinen et al. (2004b) demonstrated
that Cotesia vestalis, a specialist parasitoid of the diamondback moth (Plutella
xylostella), oriented preferably toward the scent of herbivore-damaged plants of
two white cabbage (Brassica oleracea ssp. capitata) cultivars grown at ambient
[CO2 ]. However, under elevated [CO2 ], they did not differentiate between volatiles
of intact and herbivore-damaged plants of one cultivar. These results suggest
that elevated atmospheric CO2 concentrations could weaken the plant response to
feeding by insect herbivores and thereby disturb signalling to the third trophic level.
A study with oilseed rape (Brassica napus) (Himanen et al. 2009) indicated that
lower herbivory by P. xylostella larvae reduced herbivore-induced emissions from
transgenic, insect-resistant Bt (Bacillus thuringiensis insecticidal toxin producing)
plants. However, contrary to observations in Brassica oleracea plants (Vuorinen
et al. 2004b), the study by Himanen et al. (2009) demonstrated that growth under
elevated [CO2 ] increased emissions of most herbivore-induced volatile terpenoids.
11 Multitrophic Signalling in Polluted Atmospheres 303

Furthermore, C. vestalis always oriented to host-damaged plants independent of

plant herbivore resistance or [CO2 ] suggesting that elevated [CO2 ] effects on plant
herbivore-induced BVOC emission can be plant species specific and impacts on
higher trophic levels depend on plant species and cultivar. Overall, the evidence
of elevated [CO2 ] effects on multitrophic communication in tree-based systems is
surprisingly limited, calling for future experimental work on tree model systems.

11.5.5 Interactions of [CO2 ] Elevation with [O3 ]

Polluted air in industrial areas is typically composed of a mixture of primary and

secondary pollutants. Primary pollutants, such as CO, SO2 , NOX and anthropogenic
volatile organics that are released from the actual pollution sources, including power
plants, industrial complexes and exhaust emissions from vehicles, while secondary
pollutants, such as inorganic acids (H2 SO4 , HNO3 ), ozone, and PAN (peroxyacetyl
nitrate) are formed in the atmosphere (Cape 2008). In highly polluted environments,
these pollutants are always present and act together, while in more remote areas,
ozone can be the main air pollutant affecting the ecosystems, but OH and NO3
radicals can be found in photochemically significant concentrations even in “clean”
air. Therefore, vegetation is in most cases affected by air pollutant mixtures rather
than by a single pollutant compound. Due to the dynamic process of the formation
and breakdown of secondary air pollutants, most studies assessing the effects of
pollutant mixtures on trees and their trophic interactions have concentrated on
situations where only the less reactive primary pollutants, such as CO2 and SO2 , or
end-products with longer lifetimes, such as O3, are present (e.g., Percy et al. 2002;
Vuorinen et al. 2005; Calfapietra et al. 2007). Studies concerning the direct effects
of combinations of [CO2 ] with gaseous pollutants other than O3 or gaseous pollutant
mixtures on multitrophic communication are not available to our knowledge.
Exposure of trees to elevated CO2 or O3 concentrations differentially affect
plant responses, with aboveground growth being stimulated by elevated [CO2 ]
and inhibited by elevated [O3 ] (Percy et al. 2002). Exposing deciduous trees
simultaneously to elevated CO2 and O3 concentrations can result in antagonistic
effects. Elevated [CO2 ] can stimulate stomatal closure, leading to reduced O3 flux
to the leaves and the associated cellular damage, whereas elevated [O3 ] can offset
the growth stimulation by elevated [CO2 ] (Vapaavuori et al. 2009).
Experiments at the Rhinelander free-air CO2 Enrichment (FACE) experimental
site, where deciduous trees are grown in atmospheres with either elevated CO2
or elevated O3 concentration and their combination, have provided evidence of
interactive effects of elevated [CO2 ] and oxidative pollutant [O3 ] (Percy et al.
2002; Calfapietra et al. 2007, 2013 in this volume). These observations suggest
that the impact of atmospheric pollutants on BVOC-mediated communication could
be very complicated. In addition to the degradation of herbivore-induced plant
BVOCs, oxidative air pollutants may have impacts on the behaviour of natural
enemies of herbivores by affecting the volatile signals between the herbivore and
304 J.K. Holopainen et al.

the predating carnivore or parasitoid. Aphid alarm pheromones, mostly composed of

the sesquiterpene (E)-“-farnesene, are released by aphids under attack by predators
or parasitoids. Other aphids of the same colony usually start dispersal behaviour in
order to escape potential attackers. Mondor et al. (2004) found that the dispersal
responses of the aspen aphid (Chaitophorus stevensis) to alarm pheromone released
by a mechanically damaged aphid, decreased under elevated [CO2 ], but increased
under elevated [O3 ] atmospheres when the host plant of aphids was grown at the
Rhinelander open-field exposure site. This study showed that intraspecific olfactory
communication may be radically altered in response to elevated concentrations of
different air pollutants, but intriguingly, elevated [O3 ] did not reduce the efficiency
of (E)-“-farnesene, a sesquiterpene with a relatively short lifetime at elevated [O3 ]
atmospheres. The authors did not find an explanation as to why this relatively
reactive compound induced a more pronounced response in conditions where it
should have been detected at lower concentrations. However, aphid predators such
as hoverfly (Episyrphus balteatus) larvae and lady beetles (Harmonia axyridis)
are known to eavesdrop on aphid alarm pheromones and respond in particular to
(E)-“-farnesene (Vandermoten et al. 2011). It is possible that aphid populations
could associate alarm pheromone signals with predation densities depending on
their earlier experiences with predators. Percy et al. (2002) reported very low
predator densities in elevated [O3 ] plots of the same experimental site, while
elevated [CO2 ] plots had high predator densities. Aphids adapted to high predation
rates and continuous exposure to the alarm pheromone (E)-“-farnesene, possibly
show a reduced dispersal response to alarm pheromone release by individual
aphids, because altered dispersal response does not reduce their probability of being
consumed by a predator.

11.6 Future Directions

11.6.1 Air Pollution and Volatile Signalling Compounds

in Natural Ecosystems

The need to understand the mechanisms, evolutionary role and factors affecting
BVOC-based multitrophic signalling in natural and man-made ecosystems is be-
coming more topical every day. Natural ecosystems are suffering from expanding
habitat fragmentation, which will lead to reduced biological diversity and eventually
to species extinction in small patches (Hanski 1994). Parasitoids of herbivorous
insects have different strategies in searching for populations of their host. For
example, the two primary parasitoids Hyposoter horticola and Cotesia melitaearum
of the Glanville fritillary butterfly (Melitaea cinxia) larvae have different search-
ing strategies. Hyposoter horticola moves long distances to locate herbivores in
neighbouring patches, while Cotesia melitaearum searches over short distances
and mostly stays in the same patch (van Nouhuys and Hanski 2002). In particular,
11 Multitrophic Signalling in Polluted Atmospheres 305

parasitoid species such as H. horticola relying on long distance volatile signals may
become endangered in air-polluted and fragmented environments where the BVOC
signals from herbivore-damaged plants will become weaker and possibly not reach
other distant patches.

11.6.2 Air Pollution and Volatile Signalling Compounds

in Plant Production

Exogenous elicitors, such as jasmonates and methyl salicylate are compounds

that activate various defence responses in treated plants and induce production of
secondary compounds to improve direct and indirect defence in plants (Thaler 1999;
Holopainen et al. 2009; Kaplan 2012; Semiz et al. 2012). Foraging efficiency of
parasitoids and parasitisation rates of herbivorous Lepidopteran larvae have been
increased on plants treated with elicitors over untreated control plants. This is
because of induction of emissions of parasitoid-attractive herbivore-induced BVOCs
from intact test plants treated with elicitors (Thaler 1999; Qiu et al. 2012). Elicitors
have the potential to be used in the seedling production of forest trees, although in
conifer seedlings, the response could be extensive, leading to growth of larger resin
storage organs and three orders of magnitude higher inducible monoterpene emis-
sions compared to controls (Holopainen et al. 2009). This may have consequences
for atmospheric quality by increasing ozone and secondary aerosol formation in
polluted environments and consequently, reducing the efficiency of BVOC-based
indirect plant defence under pollution episodes.
Push-pull strategies are based on the behavioural manipulation of insect pests
and their natural enemies (Cook et al. 2007). This is done by exploiting volatile
semiochemicals to repel insect pests from the crop (‘push’) and to attract them
into trap crops (‘pull’) where the pests are subsequently removed. Trap crops
could be attractive and more susceptible cultivars or cultivated separately from
the main crop (Cook et al. 2007) or used in intercropping with the main crop
(Hassanali et al. 2008). Efficiency of natural parasitoids and predators or released
biocontrol organisms to control pest species can be improved by attracting these
natural enemies by attractive BVOCs. Elicitors can be used on main crop plants, or
synthetic analogues of herbivore-induced BVOCs can be released from dispensers
embedded within the crop (Kaplan 2012). This strategy of attracting carnivorous
enemies to crops could be based on three different scenarios: (1) parasitoids are
directly attracted to released synthetic BVOCs, (2) parasitoids respond to herbivore-
induced plant volatiles that were elicited via exposure to a synthetic BVOC, or (3)
synthetic BVOCs prime neighbouring plants, and this amplifies the pest-induced
volatile production in crop plants when the crop is damaged (Kaplan 2012). Based
on our current knowledge of atmospheric behaviour, constitutively emitted species-
specific BVOCs (Himanen et al. 2010) and herbivore-induced BVOCs (Pinto et al.
2007c; Himanen et al. 2009), the push-pull strategy to repel pests from crops, e.g.,
306 J.K. Holopainen et al.

by malodorous companion plants in intercropping, is possibly more efficient in

environments with a high air pollution load than strategies to attract pest enemies
by more reactive herbivore-induced BVOCs.
The potential of semivolatile plant BVOCs which condense on plant surfaces
(Himanen et al. 2010; Karban 2010) to protect cultivated plants from herbivores is
so far a totally unexplored area of research. If these semivolatiles protect against
herbivores, what will be the impact of these compounds for natural enemies of
herbivores? The phytotoxicity of these compounds to the receiver plants (Copolovici
et al. 2005) should also be considered. Nevertheless, the emissions of Rhododendron
tomentosum that were dominated by the semivolatile oxidized sesquiterpenoids
ledol and palustrol did not show phytotoxic effects on Betula spp. (Himanen et al.
2010). Furthermore, oxidation and partitioning of herbivore-induced volatiles into
the particle phase in oxidant-rich atmospheres leads to formation of various organic
compounds including short-chain organic acids and aldehydes, and condensed long-
chain oxidized molecules. These clusters of nanoparticles composed of BVOC
reaction products can accumulate on plant surface, but it is not yet known whether
they have the potential to serve as protective repellent compounds against herbivores
(Holopainen and Gershenzon 2010). The answers to these intriguing questions may
open a way to develop new control methods against herbivores or pathogens.

11.6.3 Improved Understanding of Atmospheric Behaviour

of Herbivore-Induced BVOCs in Polluted Air

Despite the efforts in recent years, there is still a lot to learn about how atmo-
spheric pollutants affect volatile-mediated interactions. Most of the empirical data
available has been attained under laboratory conditions and these data suggest that
pollutants most likely reduce the distance over which volatile-mediated interactions
occur (Blande et al. 2010a). So far, most studies have involved fumigation with
one oxidizing pollutant, which is an experimental short-falling that needs to be
addressed in the future. Ozone, due to being both phytotoxic and reacting with
numerous plant BVOCs, has received the most attention. However, OH and NO3
radicals are more reactive than ozone with many BVOCs, including numerous
herbivore-induced plant volatiles. Thus, for better understanding of the effects of the
atmosphere on volatile-mediated interactions, empirical data need to be collected
that combine the effects of realistic combinations of oxidants at realistic proportions
and concentrations. Collection of such data will facilitate the development of models
that can better estimate the dynamics of volatile-mediated interactions under a range
of atmospheric conditions.
At present, controlled generation of OH radicals for experimental purposes
is possible and the atmospheric behaviour of herbivore-induced BVOCs can be
monitored (Hao et al. 2011). BVOC reactions with NO3 radicals are important
for nighttime emissions when NO3 radical concentrations can be sufficiently high.
11 Multitrophic Signalling in Polluted Atmospheres 307

However, the generation of NO3 radicals is currently less straightforward. Ozone

and the potentially dangerous oxidizer dinitrogen pentoxide (N2 O5 ) have been used
as precursors to produce a variety of reaction products such as pinonaldehyde and
alkylnitrates from the reaction of NO3 with ’-pinene (Wängberg et al. 1997). As
NO3 concentrations are generally very low during daylight hours, the time when the
majority of volatile-mediated interactions between herbivore damaged plants and
natural enemies of herbivores are thought to occur, a strategy involving enrichment
of OH radicals and ozone concurrently may constitute a decent interim strategy; the
long-term goal being to combine all the major atmospheric oxidants together during
experimentation.
It is further important to gain insight into the involvement of atmospheric
pollutants in altering the temporal and spatial dynamics of BVOC signalling and
how this information is interpreted by receiver organisms. It is known that the
ratios of volatile compounds in an herbivore-induced plant volatile odour plume
provide information for foraging predators and parasitoids (Pareja et al. 2009).
Small changes in those ratios can alter behavioural responses. Increases in certain
pollutants, which have different reaction times with different volatiles, can alter the
compound ratios, and thereby the informativeness for herbivores and their predators.
Such effects may occur at a much faster rate than the general degradation of the
volatile blend, and may occur at lower oxidant concentrations than generally used in
experiments. The oxidation products may also constitute signals to foraging insects
or other plants, and may provide temporal or spatial information related to the
emission source. Understanding how pollutants affect the dynamics of a volatile
signal and the plasticity of the signal-receiving organism’s response are of central
importance in understanding how atmospheric pollutants influence multitrophic
interactions.

11.7 Conclusions

To understand the full effect of atmospheric pollutants on multitrophic interactions,

we need to consider both the direct and indirect effects of the pollutants concur-
rently. The direct effects include phytotoxicity of oxidizing chemical species and
potential direct toxic effects on herbivores or their natural enemies. The indirect
effects include the degradation of volatile chemicals and the subsequent effect on
multitrophic interactions. These two facets are linked, whereby the combinations of
herbivore feeding and oxidative stress may interact to result in an induced chemical
bouquet that is different from that induced by herbivore feeding alone. The effects
of combined stresses on the volatile emissions of plants and the subsequent effects
on higher trophic levels clearly are high priority research questions for the future
studies (Holopainen and Gershenzon 2010).
In summary, the key to understanding the effects of pollutants on multitrophic
interactions lies in adopting a holistic approach that includes experimentation
with multiple co-occurring pollutants, and with consideration of the direct and
308 J.K. Holopainen et al.

indirect effects on the organisms of a multitrophic system. The effects of increased

degradation of signal compounds, role of newly created compounds, spatial and
temporal signal dynamics and system compensation through adaptation should
become the foci of future studies.

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Chapter 12
Leaf-Level Models of Constitutive
and Stress-Driven Volatile Organic
Compound Emissions

Rüdiger Grote, Russell K. Monson, and Ülo Niinemets

Abstract This chapter provides a review of past and contemporary leaf-level

emission algorithms that have been and currently are in use for modelling the
emissions of biogenic volatile organic compounds (BVOCs) from plants. The
chapter starts with a brief overview about historical efforts and elaborates on
processes that describe the direct emission responses to environmental factors such
as temperature and light. These phenomenological descriptions have been widely
and successfully used in emission models at scales ranging from the leaf to the
globe. However, while the models provide tractable mathematical functions that link
environmental drivers and emission rates, and as such can be easily incorporated in
higher scale predictive models, they do not provide the mechanistic context required
to describe interactions among drivers and indirect influences on interactions such as
those due to acclimation, accumulated stress and ontogeny. Following a discussion
of these issues and the limitations they impose on the current state of model-based
prognoses of BVOC emissions, we describe in some detail the knowledge gaps that
need to be filled in order to move BVOC emission models into forms that are more
directly coupled to physiological processes.

R. Grote ()
Institute for Meteorology and Climate Research, Atmospheric Environmental Research Division
(IMK-IFU), Karlsruhe Institute of Technology (KIT), 82467 Garmisch-Partenkirchen, Germany
e-mail: [email protected]
R.K. Monson
School of Natural Resources and the Environment and the Laboratory for Tree Ring Research,
University of Arizona, Tucson, AZ 85721, USA
Ü. Niinemets
Department of Plant Physiology, Institute of Agricultural and Environmental Sciences, Estonian
University of Life Sciences, Kreutzwaldi 1, Tartu 51014, Estonia

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 315
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 12,
© Springer ScienceCBusiness Media Dordrecht 2013
316 R. Grote et al.

12.1 Introduction

A diverse range of plant species has the capacity to emit biogenic volatile organic
compounds (BVOCs), in particular volatile isoprenoids, in a constitutive manner.
These emissions can either come from specialized storage structures, reflecting slow
vapourization and diffusion of volatiles synthesized days, weeks and months prior
to emission (“storage” emissions such as those from storage structures that play an
important role in direct plant defence), or from continuous physiological processes
(“persistent physiological emissions” such as methanol emissions from expanding
leaves). The key characteristic of constitutive emissions is that they are not
dependent on induction by external triggering factors, such as herbivory or abiotic
stress. In contrast, induced emissions result from an upregulation of key metabolic
pathways in response to external cues, thereby leading to elicitation of BVOC
emissions even in species lacking constitutive emissions. Induction of emissions,
in particular, includes the upregulation of gene expression to produce additional
enzymatic activity, e.g., the induction of genes for terpene synthases in response to
insect attack, (Litvak and Monson 1998; Arimura et al. 2000; Li et al. 2002; Babst
et al. 2009). In the case of constitutively emitted compounds, such as isoprene
and monoterpenes, environmental cues typically alter the level of expression of
key controlling enzymes (Wiberley et al. 2008, 2009), complicating separation of
stress-triggered and constitutive emission responses. The various timescales across
which emissions are influenced, and the interactions of multiple cellular processes
in determining the capacity for emissions has created challenges to describing
BVOC emissions in mathematical representations, and thus, in producing prognostic
models that reflect metabolic and physiological first principles.
In some of the processes that govern emissions we have, in fact, made progress in
linking emissions to true physico-chemical theory. For example, chemical properties
of volatiles responsible for stomatal control of emissions have been identified
(Niinemets et al. 2004; Niinemets and Reichstein 2003), and temperature depen-
dence of many BVOC emissions has been described in terms of fundamental kinetic
theory (see Grote and Niinemets 2008; Monson et al. 2012). However, the mecha-
nistic basis for alteration of BVOC emissions by growth in different light or tem-
perature regimes, the influence of drought stress, in either the short- or long-term,
and the influence of leaf ontogeny remains largely unresolved (see Monson 2013 in
this volume). Furthermore, induction of emissions following biotic or abiotic stress
events has not yet been included in emission models, although cellular signalling
models have been developed that simulate alterations in gene expression with rela-
tively good success (Vu and Vohradsky 2007; Yip et al. 2010; Muraro et al. 2012).
Apart from the importance of detailed understanding of the emission mechanisms
that determine the emissions in the timescale of seconds to minutes, the pathway
flux also depends on longer-term emission controls associated with the changes in
the capacity of the volatile synthesis pathway. Modelling seasonality has been an
especially difficult task, because seasonal variations also involve variations in light
and temperature, effects which are hard to disentangle from acclimation responses.
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 317

Furthermore, seasonal studies conducted in natural environments inevitably also

incorporate long-term stress effects such as soil drought. Embedded within these
entanglements are the summed effects of cell birth and death, both of which are
controlled by intrinsic ‘clocks’ as well as programmed responses to stress. We
know these synergies exist; we just don’t know how to represent them accurately in
mathematical representations. The mismatch between our general knowledge of the
phenomenology of the processes, and our knowledge about the stoichiometries that
determine process rates and feedbacks, has left us with little on which to base our
models. In this knowledge gap, and facing pressure from agencies and governments
to produce actionable projections of future changes in our environment, we have
produced models that work well for replicating observed emissions patterns and
dynamics. However, we also know they have limited power to be used in truly
prognostic mode, especially if the emission projections have to be made to future
conditions and to areas with limited information of species emission characteristics
and physiological performance.
In this chapter, we analyse the origin and development of both empirical
and semi-mechanistic emission model algorithms to simulate volatile emission
responses to immediate variations in empirical drivers. We try to emphasize the
gaps in knowledge that cause our projections of BVOC emissions to be bracketed
with relatively large uncertainties. Then we consider the knowledge needed to fill
some of these gaps, and incorporate especially the long-term influences, such as
acclimation mechanisms, and seasonality, in emission models. Finally, we suggest
ways to improve our representation of induced emissions in our models – that
is, how to design the models to respond to episodic forcing elements in the
environment. In assessing the conflicting states of existing knowledge and the need
for reliable projections, we end up concluding that the expansion of models to
include interactions such as acclimation and ontogeny is a valuable step forward,
as it allows for the establishment of a recognized framework within which we must
cast our projections. However, we also argue that the limitations of this approach
must be broadcast in a more amplified form. It is a dangerous situation to assume
that because a model includes a scheme for acclimation or induction, it is in a form
capable of more accurate prognosis. We emphasize that new approaches must be
developed for assessing the uncertainties created by the gap between knowledge
about the existence and basic operation of a certain process, and the exact controls
by which the model links emission rates to physiological and environmental drivers.

12.2 Modelling Environmental Dependencies

12.2.1 Brief History of Leaf-Level Emission Models

The complexity of direct emission control represented a big challenge for modelers.
Although Sanadze and Kalandaze (1966) reported the dependency of isoprene
emission rate on temperature and light long ago, it took more than a decade to
318 R. Grote et al.

produce the first mathematical relation to describe this dependency (Tingey 1979;
Tingey et al. 1981). In developing this first mathematical model, it was noted that
isoprene emission in studied broad-leaved species responded to temperature as well
as to light, while monoterpene emissions in studied conifers only responded to
temperature (Tingey et al. 1980). Thus, monoterpene emission was assumed to
originate solely from storage pools within the plant (e.g., oleoresin) that provide
an unconstrained source, at least in the case of emissions over minutes to hours to
days. Accordingly, most of the control over the short-term monoterpene emission
rates was relegated to diffusive resistances. The mechanism of isoprene production
was clarified through the studies of Monson and Fall (1989) who highlighted the
relationship to photosynthesis (see also Loreto and Sharkey 1990; Monson et al.
1992, 1994). Recognition of this relationship allowed Guenther et al. (1991, 1993)
to develop models for leaf-scale isoprene and monoterpene emissions based on the
shapes of the light and temperature response curves previously used in photosyn-
thesis models (e.g., Farquhar and von Caemmerer 1982; Tenhunen et al. 1976).
Both photosynthesis as well as isoprene emission show an optimum relationship to
temperature and a saturation response to increasing light. The temperature optimum
of BVOC emission is, however, high compared to most physiological processes
(Fig. 12.1a, b). In contrast, the light dependency of isoprene emission is similar to
that of photosynthesis (Fig. 12.2a, b). Thus, while the metabolic linkages between
photosynthesis and isoprene emission were clear, there was also evidence that
isoprene biosynthesis has a unique set of controlling processes that could not be
ignored. Later, it was recognized that some species emit monoterpenes that are
tightly coupled to their biosynthesis in the chloroplast, and thus, the monoterpene
emission mechanisms in these species are similar to isoprene emission mechanisms
(Schürmann et al. 1993; Staudt and Seufert 1995). Shortly after the first compre-
hensive emission models were presented, the main biosynthetic pathway of volatile
isoprenoid production in plant plastids, 2-C-methyl-D-erythritol 4-phosphate/1-
deoxy-D-xylulose 5-phosphate pathway (MEP/DOXP pathway), was discovered
(Lichtenthaler et al. 1997; Rohmer et al. 1993; Eisenreich et al. 2001). Given the
central role of the MEP/DOXP pathway, the following efforts of mechanistic and
semi-mechanistic BVOC emission model development have primarily focused on
understanding linkages to photosynthesis and controls over kinetics within this
pathway (Niinemets et al. 1999; Martin et al. 2000; Zimmer et al. 2000).
Shortly after the first leaf-scale models were published, global-scale modelers
began to incorporate some of the schemes into projections at scales with con-
siderably longer time and greater space intervals (e.g., Guenther et al. 1995).
These projections were inherently constrained by the bottom-up approach, because
in this framework there was little potential to validate model predictions. Even
within the context of atmospheric chemistry, large gaps in our knowledge, for
example of the degree of molecular oxidation of isoprene and the deposition of
oxidation products, precluded validation of projected global emission rates. Despite
acknowledgement of large uncertainties, the models continued to be expanded in
terms of the processes they included (e.g., Fuentes and Wang 1999; Guenther et al.
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 319

Fig. 12.1 Comparison of

shapes of temperature
responses of isoprene and
monoterpene emissions in
species lacking specialized
storage structures (a, b), and
monoterpene emissions in
species with terpene storage
structures (c). Panel (a)
highlights the differences
between the various versions
of the Guenther isoprene
emission algorithm presented
in 1991, 1993, and 1999
(G91, G93, and G99),
(b) compares different
monoterpene emission
parameterizations in the
broad-leaved evergreen
sclerophyll (Quercus ilex),
while (c) compares the
suggested temperature
responses among species
(Tingey et al. 1980; Guenther
et al. 1991; Holzinger et al.
2006; Ruuskanen et al. 2005;
Hakola et al. 1998, 2003;
Owen et al. 1997). The
broken lines correspond to
Eq. 12.2 and continuous lines
to Eq. 12.3

2000; Müller et al. 2008; Lavoir et al. 2011; Ashworth et al. 2013; Guenther 2013).
For example, empirical relationships linking isoprene emission rate to atmospheric
CO2 concentrations (Rosenstiel et al. 2003; Wilkinson et al. 2009) were included
in existing global emission models (Arneth et al. 2007; Heald et al. 2009). The
‘Model of Emissions of Gases and Aerosols from Nature’ (MEGAN) (Guenther
et al. 2006, 2012) is currently the most widely used model for projecting global
trends in BVOC emissions. MEGAN includes some of the more difficult long-term
influences on emissions, such as temperature acclimation, response to drought and
320 R. Grote et al.

a
1.2

Relative emission response

1.0

0.8

0.6
F. sylvatica
0.4 Q. alba

0.2 Q. ilex (monoterpenes)

G93 normalized
0.0
b
1.2
Relative emission response

1.0

0.8

0.6

0.4 Q. alba, sun

Q. alba, shade
0.2
G93 normalized
0.0
0 500 1000 1500
Photosynthetic quantum flux density (mmol m−2 s−1)

Fig. 12.2 Shapes of light responses of the emissions of isoprene and monoterpenes. Panel (a)
compares the light response for temperate broad-leaved deciduous trees Fagus sylvatica (Schuh
et al. 1997), and Quercus alba (sun foliage, Harley et al. 2007), and Mediterranean evergreen
sclerophyll Q. ilex (Lavoir et al. 2011) along with the G93 (Guenther et al. 1993) estimate, while
(b) shows the light responses for different canopy layers in Q. alba from Harley et al. (1997)

ontogeny. However, these schemes are largely non-validated, except for a few case
studies. Not yet considered in BVOC emission models are responses to herbivory
and pathogen infection, oxidative air pollution stress and soil infertility (Loreto
and Schnitzler 2010) and priming of emissions by consecutive and simultaneous
stresses or mild stress episode(s) preceding a more severe stress (Niinemets
2010a, b).
One of the more promising approaches to emerge in the past decade with
regard to validating model projections and reducing uncertainties is the fusion of
satellite remote sensing of formaldehyde and glyoxyl (oxidative products of terpene
BVOCs; see Abbot et al. 2003; Palmer et al. 2003, 2006; Ashworth et al. 2013)
with emissions models. Inverse modelling of formaldehyde and glyoxyl columns to
reveal the locations and magnitudes of BVOC sources and sinks has the potential
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 321

to provide new insight into time-dependent interactions between emissions and

environmental change, especially at the scales needed to integrate processes from
single leaves to regional ecosystems.
In the following sections, we discuss how different environmental drivers are
represented in contemporary emission models. Most of these models follow the
general idea that there is a certain capacity for the emission of a given compound
that depends on the overall diffusion resistance (“storage” emissions) or by the
rate of volatile formation (“instantaneous” emissions). The emission capacity can
be defined as the maximum emission rate that is physiologically possible (EMAX ).
For “instantaneous” emissions, this is typically observed at saturating light and a
temperature of about 40 ı C (see Fig. 12.1a, b; Fig. 12.2). However, no such apparent
physiological limitation exists for storage emissions, which are driven solely by
volatilization and diffusive resistance. Thus, in emission studies, one often uses
a standardized emission rate, ES (also called the emission factor) that is defined
as the emission rate under certain arbitrarily selected environmental conditions
(Guenther et al. 1991; Niinemets et al. 2010c). The standard conditions are typically
set as the leaf temperature of 30 ı C (303.16 K), incident quantum flux density of
1,000 mol m2 s1 (Guenther et al. 1991, 1993) and ambient CO2 concentration
of 400 mol mol1 (Wilkinson et al. 2009). Following Guenther et al. (1991), the
general form of this approach to estimate the emission rate of a specific compound
or compound group, E, can be expressed as:

E D ES f .I1 / f .I2 / : : : f .In / ; (12.1)

where f (Ii ) represents different response functions (i D 1..n) scaling ES to en-

vironmental conditions other than the standard conditions. These functions are
addressed separately in the following paragraphs. Implicit in Eq. 12.1 is that
different environmental factors affect the emission rate independently. This is not
necessarily valid (e.g., Sun et al. 2012) and will be addressed afterward together
with alternative ways of estimating E without the need to specify ES .

12.2.2 Temperature Dependency

BVOC emissions can originate from specific storages such as resin ducts, or
from organelles that are not specifically formed to hold BVOCs (e.g., isoprene
from chloroplasts, methanol from cell walls or sesquiterpenes from the cytosol).
In some cases, the temperature dependency results from the pathway producing
the given compound being sensitive to temperature (here, referring to short-term
dynamics in temperature). In other cases, it is the temperature dependency of
compound volatility that most determines the emission rate. The first type of
temperature dependency is exemplified by the emission of isoprene, methylbutenol,
and light-dependent monoterpenes. The second type of temperature dependency is
exemplified by the emissions of stored compounds, like the monoterpenes emitted
from the resin systems of many conifers. Thus, two separate modelling strategies
are developed to represent both types of emission.
322 R. Grote et al.

12.2.2.1 Temperature Effects on Storage Emissions

These emissions have been described by Tingey et al. (1980) by fitting emissions on
a log scale to temperature using a linear relationship:

f .T / D exp Œp1  T C p2  (12.2)

with T representing air temperature and p1 and p2 being empirical parameters.

Guenther et al. (1993) used the following exponential relationship to temperature:

f .T / D exp Œˇ .TL  TS / ; (12.3)

where TL is the leaf temperature and TS is the reference temperature (set to

303.16 K D 30 ı C) and ˇ is an empirical coefficient. Guenther et al. (1993) set ˇ to
0.09 (K1 ) based on observations using 28 species. It should be noted that species-
specific estimates ranged from 0.057 to 0.148 in the original Guenther et al. (1993)
study, a range that has since been exceeded in other measurements. For example
Pokorska et al. (2012) estimated ˇ to be 0.24 for Abies alba trees in late summer and
Owen et al. (1997) found values larger than 0.3 for Cistus incanus plants (see also
Fig. 12.1c). In addition, Helmig et al. (2007) showed that ˇ also changes within the
canopy. Equations 12.2 and 12.3 have been widely used to describe emissions from
storage pools, including those for monoterpenes and sesquiterpenes (e.g., Ormeño
et al. 2010).
The implicit assumption in Eq. 12.3 is that the resistance between different
types of storage systems and the air is constant. This implies that the storage
size is large relative to the emission rate and is not depleted during the emission
period – an assumption that has been challenged by the work of Schurgers et al.
(2009) who stated that storage emissions in a Pinus ponderosa forest could best be
described considering a dynamic change in leaf monoterpene concentration during
the year.

12.2.2.2 Influence of Temperature on Immediate Emissions

The emissions that are tightly coupled to production, increase with increasing
temperature in an exponential fashion up to a maximum rate, thereafter the
emissions decrease rapidly, reflecting enzymatic degradation or substrate limitations
(e.g., Monson and Fall 1989; Loreto and Sharkey 1990; Monson et al. 1992; Rasulov
et al. 2010, 2011). Based on earlier work that relied on chemical kinetics theory such
a relation has been postulated by Johnson et al. (1942) to take the form:
h i
exp cT  H RTL
A

f .T / D h i (12.4)
1 C exp R  H
S
RTL
D
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 323

where HA is enthalpy of activation in J mol1 , HD is the enthalpy of de-activation in

J mol1 , S is an entropy term and R is the universal gas constant both in J K1 mol1 ,
and cT is a scaling constant. Following the form of this relationship, but substituting
the enthalpy and entropy terms with empirically derived parameters, Guenther et al.
(1991) rewrote the equation as:
h i
.TL TS /
exp cT1RT L TS
f .T / D h i (12.5)
cT2 .TL TM /
1 C exp RTL TS

where cT1 (J mol1 ), cT2 (J mol1 ) and TM (K) are ‘tunable’ coefficients. Guenther
et al. (1999) modified the form of Eq. 12.5 to refer directly to the temperature
optimum Topt (K) rather than to TS :

exp .cT4 x/
f .T / D cT 3 (12.6)
cT3  cT4 .1  exp .cT3 x//
 
1=Topt  .1=TL /
where x D
R
The parameters cT3 and cT4 (both in J mol1 ) are again empirically determined coef-
ficients. Mathematically, Eqs. 12.5 and 12.6 are equivalent to Eq. 12.4, considering
that some differences are absorbed within the coefficients (see Monson et al. 2012).
Equation 12.4 was also applied in the models of Niinemets et al. (1999) and Mar-
tin et al. (2000) for describing the temperature response of isoprene synthase and
Niinemets et al. (2002c) for describing the temperature response of monoterpene
synthases. Zimmer et al. (2000) used it to characterize the temperature dependency
of isoprene formation from precursor substances. In later work, the Zimmer et al.
(2000) model was applied to other isoprenoids using the same temperature response,
but with parameters determined independently for various processes within the
MEP pathway (Grote et al. 2006). In their studies, these parameters were derived
through an inverse modelling approach whereby the ‘best-fit’ parameter values were
determined after assimilating enzyme temperature dependencies into the model.
The temperature dependence of isoprene synthase activity was based on in vitro
estimations of synthase activity in crude leaf extracts of Populus tremuloides
(Monson et al. 1992) and Quercus robur (Lehning et al. 1999), and for monoterpene
synthase extracts of Quercus ilex (Fischbach et al. 2000).

12.2.3 Light Dependency

The original Guenther et al. (1991) model was developed on the basis of numerous
studies of Sanadze (1964), Tingey et al. (1981), Monson and Fall (1989) and Loreto
and Sharkey (1990) that indicated a functional linkage between photosynthetic
CO2 assimilation and the formation of some BVOCs, especially isoprene. It had
324 R. Grote et al.

been apparent that absorbed photon flux density is the principal driver for this
similarity, suggesting that both processes occurred in the chloroplast, both processes
exhibited similar shapes in their light-response curves, and both processes required
the input of reductant from the photosynthetic electron transport system. The light
dependency of the thylakoid electron transport rate (J, mol m2 s1 ) can be
described mathematically as:

J D 0:5 a I .1  b/ (12.7)

where a is the fraction of incident photosynthetic photon flux density (I in

mol m2 s1 ) absorbed by the leaf, and b is the fraction of the absorbed photon
flux diverted to processes other than photosynthetic electron transport. Implicit in
Eq. 12.7 is that J is not saturated by the absorbed I. As electron transport starts
to become light-saturated with increasing light intensity, the dependence of J on
I will begin to exhibit a curvilinear shape. Recognizing that J is influenced by an
upper limit (Jmax , mol m2 s1 ), and recognizing that the influence of Jmax on J
increases as I increases, the following quadratic equation has been developed for
photosynthesis:

0 D J 2  Œ0:5 a I .1  b/ C Jmax C  J C 0:5 a I Jmax .1  b/ (12.8)

where  is a tunable ‘curvature factor’ that theoretically can vary from 0

(rectangular hyperbola) to 1 (Blackman type response) with a default value of
0.85 corresponding to moderate curvature. Equation 12.8 describes a rectangular
hyperbola in which a continuous transition occurs from J D 0 at I D 0 to J D Jmax at
saturating I (Farquhar and Wong 1984).
Guenther et al. (1991) used Eq. 12.8 to develop an analogue model to describe
the light dependency of isoprene emission (as a multiplication factor for Eq. 12.1):
p
x x 2  4 b aI cL1
f .I / D (12.9)
2 cL1

where x D b aI C cL1 C cL2 :

The parameters cL1 and cL2 are tunable coefficients that account for the differ-
ences in molar stoichiometry of electron transport between isoprene formation and
CO2 and reflect the curvature coefficient () and the upper limit of the function
formerly defined by Jmax . Note that the meaning of the coefficients a and b is
also different for the isoprene light response than for the light response of J.
In later publications, Guenther et al. (1993) used a new form for J, aligning it
with a mathematical expression of the so-called Smith’s (Smith 1937) equation of
photosynthesis (see Tenhunen et al. 1976; Harley et al. 1992):
˛I
J D r  (12.10)
2 2
1 C J˛maxI 2
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 325

where ˛ is the initial slope of the response carrying the units of mol mol1
photons incident to the leaf (the apparent quantum yield). Equation 12.10 defines
the shape of a rectangular hyperbola that approaches an asymptote at relatively
high values of I. Adopted from this expression Guenther et al. (1993) described
the light dependency of BVOC (isoprene) emission by removing Jmax and adjusting
the function with an additional parameter (cL3 ). However, as Monson et al. (2012)
pointed out, there was a mathematical violation in the denominator in that the square
root quantity contains a sum that mixes a unitless constant (1.0) with the product of
two terms (˛ and I) that carry units. Thus the equation should be written as:
˛ISO cL3 I
f .I / D s  (12.11)
˛2 I 2
1 C ISO2
cx

where cL3 is now in m2 s mol1 , and ˛ ISO now carries units mol mol1 photons
incident to the leaf, while cx is in mol m2 s1 . It can be set to 1.0 to represent
the same response as before. The coefficients for ˛ ISO and cL3 in Eq. 12.11 were
assumed to be constant in the Guenther et al. (1993) analysis, but it was later
realized that they can vary within the canopy (Fig. 12.2), partly reflecting the explicit
connection between the emission capacity and ˛ ISO (Monson et al. 2012), and partly
reflecting acclimation within the canopy (Sect. 12.3.2).
Niinemets et al. (1999) followed a different path and related the emission rate to
light using an explicit connection with J. They have therefore used an expression
of J limited by ribulose-1.5-bisphosphate (RuBP) regeneration related to net CO2
assimilation, A, mol m2 s1 :

.A C RD / .4 Ci C 8   /
J D (12.12)
Ci   
where Ci is the CO2 mole fraction in the intercellular air spaces of the leaf, RD is
the rate of non-photorespiratory respiration rate in light (mol m2 s1 ) (mostly
mitochondrial respiration), and  * is the hypothetical CO2 compensation point
in the absence of RD that depends on Rubisco kinetic characteristics. Using this
relation, Niinemets et al. (1999) linked the emission rate (E) to photosynthetic
electron transport rate. However, expressing J from Eq. 12.12 only yields the rate
of photosynthetic electron transport that is needed to reduce CO2 to the level of
immediate photosynthetic product, sugars, and that needed for photorespiration. As
isoprene is a more reduced molecule than sugars, additional reductive equivalents
are needed to synthesize isoprene. Including this additional electron transport rate,
the rate of isoprene emission, Eiso , was expressed as (Niinemets et al. 1999):

.Ci    /
Eiso D " JT (12.13)
6 .4:67 Ci C 9:33  /

where JT is the total electron transport rate, i.e., that used for CO2 fixation and
photorespiration plus that needed for additional reduction of sugars to the level of
326 R. Grote et al.

Fig. 12.3 Comparison of a

measured and simulated light 20
dependencies of isoprene
emission rate (a), ’-pinene

−2 −1
15
(b) and total monoterpene
emission rate (c) in
broad-leaved deciduous tree 10 r2
Liquidambar styraciflua (a),
and in evergreen sclerophyll
5
Quercus ilex (b, c). In (a), the Measured
measurements were Simulated
conducted at a constant leaf 0
temperature of 25 ı C (Data 0 500 1000 1500 2000
from Niinemets et al. 1999), b
3
in (b) at 30 ı C (Data from
Loreto et al. 1996), and in (c),

−2 −1
the data were filtered from
daily time-courses of 2
monoterpene emission
measured between Aug. 3–5,
1994, for a temperature range 1
of 25–35 ı C (Niinemets et al.
2002c). The data were fitted
r2
by Niinemets et al. (1999,
0
2002c) isoprenoid emission
model (Eq. 12.14). Only one
c
15
leaf-dependent coefficient, ©,
the fraction of electrons in
−2 −1

isoprenoid synthesis, was

10
used in (a), and (b), while the
dependence of © on leaf
temperature was considered
in (c) using an exponential 5
scaling coefficient (a£ a
(Niinemets et al. 1999, r2
2002c) 0
0 500 1000 1500
−2 −1

isoprene, and © is the fraction of JT required to synthesize isoprene. The dependence

of J on I was modelled using Eq. 12.10 and the resultant value of J was inserted
into Eq. 12.13. In this equation, the connection between isoprene emission and pho-
tosynthetic electron transport results from the assumption that NADPH availability
controls the rate of isoprene biosynthesis, although an analogous dependence on
ATP availability has also been formulated (Niinemets et al. 1999). Thus, the light
dependence of isoprene emission could be explained by only one isoprene synthesis-
specific coefficient, © (Fig. 12.3a). In further model development (Niinemets et al.
2002c; Niinemets 2004), the equation was generalized as:

.Ci    /
E D " JT ; (12.14)
& .4Ci C 8  / C 2 .Ci    / .#  2&/
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 327

where − is the carbon cost of isoprenoid emission (6 mol mol1 for isoprene and
12 mol mol1 for monoterpenes), and # is the NADPH cost of specific isoprenoid
compounds (mol mol1 ). Differently from the initial model formulation (Niinemets
et al. 1999) where the extra electron transport was assumed to originate from
mitochondrial catabolism of the photosynthetically fixed carbon, Eq. 12.14 assumes
that the rate of photosynthetic electron transport in photosynthezising leaves is
larger than can be predicted by Eq. 12.12, and thus, the extra reductive equivalents
rely on this “excess” electron transport (Niinemets et al. 2002c; Niinemets 2004).
Overall, the model fit to monoterpene emissions was good (Fig. 12.3b, c), although
it was realized that less volatile terpenoids can be non-specifically stored in the
leaf liquid and lipid phases, resulting in delays between biosynthesis and emission
(Niinemets et al. 2002a, c; Niinemets and Reichstein 2002).
Zimmer et al. (2000) and Grote et al. (2006) modelled BVOC emission on the
basis of changes in metabolite pools. Their numerical model is based on reaction
rates of various enzymes in the production pathway that are described based
on Michaelis-Menten kinetics. Dynamics in the concentration of photosynthates
that also serve as primary emission precursors, pyruvate and glyceraldehyde 3-
phosphate, are directly related to photosynthesis and then enter the isoprenoid
synthesis pathway as substrates. Thus, in this form of model logic, the light
dependencies of isoprene and light-dependent monoterpene emission are introduced
by the photosynthesis model used to produce emission substrates. Thus, the light
relationship is ultimately driven by the same dependence of J on I that was reflected
in the Guenther et al. (1991) and Niinemets et al. (1999) models.
As a further simplification, Niinemets et al. (2013 in this volume) directly linked
isoprene emission to photosynthesis. In the so-called C-ratio model, isoprenoid
emission was calculated as the product of the gross assimilation rate and monoter-
pene emission to assimilation rate ratio, rC (Niinemets et al. 2013 in this volume).
While being the simplest model, rC was shown to depend on light and temperature,
and thus, required somewhat greater parameterization effort than linking emissions
to electron transport rate (Eq. 12.14). Nevertheless, comparison of different model
approaches (Eqs. 12.11 and 12.14 and C-ratio model) at canopy level indicated that
once correctly parameterized, all models performed similarly (Niinemets et al. 2013
in this volume).
As Monson et al. (2012) pointed out, all these approaches share a com-
mon ‘quasi-mechanistic’ basis in their relation to photosynthesis, with ‘quasi-
mechanistic’ meaning that the dependence is not yet fully understood so that
uncertainties remain. Although the overall flux of electrons going into volatile
isoprenoid synthesis is small, there is evidence of control of the MEP pathway flux
by ATP and/or NADPH status of chloroplasts (Loreto and Sharkey 1993; Rasulov
et al. 2009, 2011; Li and Sharkey 2013a, b). This might suggest that the effective
Michaelis-Menten constant of MEP pathway for ATP and/or NADPH controls the
pathway flux. Given that daytime production of reductant and chemical energy in
the chloroplast is driven by J, linking isoprenoid biosynthesis to the assimilation of
CO2 and to the supplies of precursor molecules into the MEP pathway constitutes
still a promising way to simultaneously model CO2 exchange and volatile isoprenoid
emission.
328 R. Grote et al.

12.2.4 CO2 Responses

The negative relationship between isoprene emission and the concentration of

atmospheric CO2 was first reported in Sanadze (1964). The response of emission
rate to changes in intercellular CO2 concentration (Ci ) follows a pattern with an
optimum at a Ci of 150–200 mol mol1 (Ci,opt ) (Loreto and Sharkey 1990; Rasulov
et al. 2009; Sun et al. 2012). It has been demonstrated that the CO2 dependence
of isoprene emission is determined by the immediate precursor, dimethylallyl
diphosphate (DMADP) pool size over the whole CO2 range (Rasulov et al. 2009;
Sun et al. 2012), but there is still a debate as to why DMADP pool size varies with
CO2 concentration.
Most of the modelling efforts have focused on understanding the decline in iso-
prene emission at CO2 concentrations exceeding Ci,opt . Sanadze (2004) developed
a biochemical hypothesis to explain this decline by postulating a competitive parti-
tioning of chloroplast reductant and energy between the reductive pentose phosphate
pathway and the MEP pathway. According to the hypothesis, the partitioning in
turn depended on the demand for reductant and energy by the reductive pentose
phosphate pathway. If this is low, these compounds are more readily available for
isoprenoid production, implying that the pathway under these circumstances acts as
a kind of excretion system. Conversely, under conditions of high Rubisco activity
(e.g., high Ci ), the reductant and energy will be diverted predominantly to synthesize
and process sugars from photosynthesis.
This logic was to some degree already captured in the model developed by
Niinemets et al. (1999) which is based on photosynthetic electron transport rate
with isoprene biosynthesis rate defined by the fraction of J that is partitioned to the
MEP pathway (Eq. 12.13). In subsequent work, therefore, based on observations,
Arneth et al. (2007) introduced an additional empirical relation into Eq. 12.13
characterizing the partitioning as a hyperbolic function of Ci . Following this same
concept, the model produced by Martin et al. (2000) represented isoprene emission
as driven by competitive partitioning of chemical energy. In this model, as Ci
increased, a negative feedback loop was imposed on emission by the decreasing
availability of ATP.
On the other hand, experiments by Rosenstiel and others (Rosenstiel et al. 2003,
2004; Loreto et al. 2007) have suggested that the CO2 sensitivity of isoprene
emission can also be explained by competition for carbon substrate between
cytosolic and chloroplastic processes. Wilkinson et al. (2009) model is based
on this proposed mechanism and follows the assumptions that (1) at low Ci ,
the availability of recently-produced photosynthates limits isoprene production,
although carbohydrate reserves may allow for some emission, and (2) that at
increasing Ci , enzyme activity limits isoprene biosynthesis, while carbon precursors
are getting more adequate. The overall response to Ci can then be expressed with an
inverse sigmoidal function:

EMAX .Ci /cC1
f .C / D 1  (12.15)
.C  /cC1 C .Ci /cC1
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 329

C* is a reference Ci and cC1 is a unitless scaling coefficient that forces the right-hand
term to be reduced exponentially at low Ci and increase exponentially at high Ci .
A similar model that is based on the concentration of dimethylallyl diphosphate,
[DMADP] in the chloroplast rather than Ci has been proposed by Possell and
Hewitt (2011). Because [DMADP] decreases as Ci increases, in those cases where
a negative CO2 response has been observed, the model takes the following form:

VMAX ŒDMADPcC 2
f .C / D (12.16)
KM cC 2 C ŒDMADPcC 2

where VMAX and KM are the Michaelis-Menten constants for isoprene synthase,
and cC2 is a unitless scaling coefficient. This model was shown to provide good
descriptions of the CO2 response for several species. However, we note that
no model is currently able to mechanistically capture the reduction of isoprene
emission at CO2 concentrations below Ci,opt of ca. 150–200 mol mol1 (Loreto
and Sharkey 1990; Rasulov et al. 2009; Sun et al. 2012), and empirical fits best
describing the entire CO2 response curve of isoprene emission have been suggested
(Sun et al. 2012).
Very recently, Harrison et al. (2013) proposed another relation of isoprene
emission to photosynthesis, assuming that isoprene emission depends on excess
reducing power, which is increased by the electron transport supply (J), and reduced
by the electron transport requirements for the dark reactions of photosynthesis.
The excess or deficit of electrons produced by photochemical reactions during
photosynthesis can be calculated as the difference between the total photosynthetic
electron flux and the total flux of electrons used for carbon assimilation that is
determined by Ci , J and maximum carboxylase activity of Rubisco (Vc,max ). The
isoprene emission rate is thus given by:

ŒCi C 2  
Eiso D p3 J  p4 Vc;max (12.17)
ŒCi C Kc;M 

where p3 and p4 are empirical parameters that represent the ‘baseline’ fraction of the
total photosynthetic electron flux used for isoprene synthesis (p3 ), and the fraction
of ‘excess’ electron flux (i.e., electrons not used in photosynthetic carbon fixation)
used for isoprene synthesis (p4 ), and Kc,M is the effective Michaelis-Menten constant
for Rubisco carboxylase activity. The approach is attractive, combining CO2 and all
other direct effects on photosynthesis, but it remains to be validated by mechanistic
knowledge concerning the relation of J to Eiso , and it does not fully address the
combination of both substrate availability and isoprene synthase activity as controls
over Eiso .
Sensitivity of BVOC emission to the atmospheric CO2 concentration has, to this
point, been described only for isoprene. Yet, we know that the substrate constraints
and mechanisms that affect the MEP pathway should affect the production of other
terpenoid compounds as well. Thus, in species without specialized terpene storage
structures, analogous CO2 -responsiveness of monoterpene emissions is expected.
Indeed, a decrease in the rate of monoterpene emissions with increasing CO2
330 R. Grote et al.

concentration has been found in Quercus ilex (Loreto et al. 2001) and (to a smaller
degree) in Betula pendula (Vuorinen et al. 2005). In other studies, no effects (Baraldi
et al. 2004; Paoletti et al. 2007) or even an increase of monoterpene emission (Staudt
et al. 2001; Himanen et al. 2009) have been observed. Clearly more work is needed
to gain insight into CO2 effects on monoterpene emissions, and it remains to be
tested if and under which conditions the described models are applicable for direct
emissions other than isoprene. Given the large number of terpene synthases and
highlighted differences in regulation for some of these synthases (Rajabi Memari
et al. 2013; Rosenkranz and Schnitzler 2013), simulating monoterpene emissions
under future conditions is currently bound to large uncertainties.

12.2.5 Needs for Future Developments

Implicit in constructing the isoprene emission model as a product of multiplicative

type equations (Eq. 12.1), is that environmental drivers such as light, temperature
and CO2 independently affect isoprene emission, i.e., any response function does
not depend on other response functions. Recent progress in determining the
mechanistic underpinnings of isoprene emission defines DMADP concentration and
kinetic controls over isoprene synthase activity as basic determinants. DMADP
concentration depends on energy/reductant availability, as well as on temperature
and photosynthetic precursors, while the kinetic controls over isoprene synthase
activity depend on temperature and DMADP concentration (Monson 2013). How-
ever, mixed control by both factors has not yet been fully reflected in models.
For example, recent research indicates that the temperature response of isoprene
emission depends on DMADP concentrations only at temperatures greater than
30 ı C (Magel et al. 2006; Rasulov et al. 2010; Li et al. 2011). The situation
is analogous with the CO2 response. Given that DMADP availability ultimately
controls the whole CO2 response of isoprene emission, and DMADP level is also
affected by light availability (Rasulov et al. 2009), CO2 responses can vary in
their dependence on the instantaneous photosynthetic photon flux density. Such
a modification in the shape of CO2 -response curve by light has been recently
demonstrated by Sun et al. (2012). The interactive effects of key environmental
drivers suggests that models based on DMADP pool size may be more accurate
for simulating isoprene emissions under co-varying light, temperature and CO2
conditions.
The models that have been based on cytosol-chloroplast competition for substrate
have not been able to explain one aspect of the CO2 response – the steep reduction
toward zero of the isoprene emission rate at a critically low value of Ci (Loreto
and Sharkey 1990; Rasulov et al. 2009, 2011; Sun et al. 2012). Typically, this value
is 150–200 mol mol1 that may be occasionally reached under drought in leaves
in their native environments. Furthermore, the declining part of the CO2 response
curve below this critical threshold can provide fundamental information on the
mechanism(s) responsible for the overall CO2 dependence of isoprene emission,
and clearly, this is an issue in need of further study.
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 331

Rasulov et al. (2009) used observations of the response of Eiso and DMADP pool
size as a function of Ci to argue that the CO2 effect on Eiso is due to variations
in chloroplastic ATP supply, not variations in the channeling of PEP from the
cytosol to the chloroplast. Both hypotheses rely on the fundamental observation
that plastidic DMADP pool size decreases as Ci increases; the debate posed by
Rasulov et al. (2009), as a counterpoint to the perspective of Rosenstiel et al. (2004),
is focused on the cause of that decrease. Most of the evidence underlying both
perspectives is correlative – positive correlations between ATP availability and Eiso
have been observed (Loreto and Sharkey 1993) and negative correlations between
PEP carboxylase activity and Eiso have been observed (Rosenstiel et al. 2003, 2004;
Loreto et al. 2007; Possell and Hewitt 2011). In a recent study by Trowbridge
et al. (2012), proton-transfer reaction mass spectrometry was used to detect the
differential kinetics of 13 C incorporation into fragments of isoprene presumed to
come from cytosolic versus chloroplastic sources. The results during periods of low
versus high Ci suggested slower labelling in the fragment originating from cytosolic
sources, and this fragment was more highly labeled in the presence of low CO2 ,
compared to that derived directly from glyceraldehyde 3-phosphate (GAP). These
latter results might be used as support for the Rosenstiel et al. (2003) perspective.
Once again, this is an issue that needs more study before a definitive model for Ci
can be identified.

12.3 Modelling Acclimation and Seasonality

Seasonal dynamics of physiological pre-conditioning have long been either ne-

glected (particularly when only short periods have been investigated) or have been
empirically adjusted to time series measurements (e.g., Staudt et al. 2000, 2002).
However, instantaneous emission responses to environmental drivers and maximum
emission rates depend on the weather conditions days to weeks prior to the emission
measurements and on ontogenetic changes in foliage emission capacity. That is why
recent weather as an important driver of isoprenoid emission rate is now increasingly
included in emission models (Guenther et al. 2006; Keenan et al. 2009; Niinemets
et al. 2010a).

12.3.1 Seasonal Changes, Leaf Age Effects and Temperature

Acclimation

12.3.1.1 Empirical Dependencies

After establishing that plants emit isoprenoid compounds instantaneously in a

manner that is dependent on light and temperature, it was recognized that these
dependencies change during the season (Ohta 1986). This has been noted to
332 R. Grote et al.

lead to considerable biases in total emission inventories and has been related to
temperature degree sums in past studies, similar to the metric used to describe
phenological development in plants (Monson et al. 1994). Nevertheless, most early
attempts on seasonal adjustment related the emission capacity to the day of the
year (D). Schnitzler et al. (1997) proposed an asymmetric equation to define the
seasonal factor, f (S), which was intended as an additional multiplier in Eq. 12.1,
and was described by an equation analogous to those used for enzyme activity
modelling:

exp .cS1 D C cS2 /

f .S / D (12.18)
1 C exp .cS3 D C cS4 /

where cS1n are curve fitting coefficients. Pier and McDuffie (1997) used a second-
order polynomial with three parameters to describe symmetric seasonal variation of
isoprene emission potential observed in white oak (Quercus alba):

f .S / D cS5 C cS6 D C cS7 D 2 (12.19)

Another equation that included parameters with physical meaning was proposed by
Staudt et al. (2000) describing a Gaussian (bell-shaped) response with an offset:
h  i
f .S / D 1  1  exp .D  D0 /2 = (12.20)

with representing the relative annual amplitude of the maximum possible seasonal
emission rate (between 0 and 1.0), D0 the day at which the emission capacity reaches
a maximum, and £ the breadth (kurtosis) of the seasonal amplitude in days. Addi-
tional asymmetric functions have been used by Lavoir et al. (2011), Keenan et al.
(2009) and Niinemets et al. (2013 in this volume). Keenan et al. (2009) compared
the seasonality function shapes, asymmetric vs. symmetric, and concluded that an
asymmetric function better adheres to the data and is recommended for simulation
of seasonal variations in isoprenoid emission.

12.3.1.2 Dependencies Imposing Genetic and Environmental Controls

Early in the history of isoprene emissions studies, it was hypothesized that it is

not the day of the year, but the previous integrated environmental conditions that
determine seasonal shifts in the isoprene emission rate (Monson et al. 1994). As
a consequence, it has been proposed that leaf developmental processes, controlled
by genetic-environment interactions, underlie expression of the genes for emission
synthases. Two modelling approaches were suggested: (1) isoprene synthase devel-
opment follows leaf phenology, assuming that only fully-grown and active leaves are
able to emit BVOCs at potential rates; (2) synthase activity is subject to continuous
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 333

but slow formation and decay processes that depend on environment. Thus, previous
environmental conditions are important determinants of Emax . Lehning et al. (2001)
followed this concept explicitly. The Seasonal Isoprenoid synthase Model (SIM)
is split into a description of leaf development and senescence, and an equation that
calculates dynamics of enzyme activity. The first mechanism represents the building
and decline of emission capacity assuming a linear relation to leaf development (or
more precisely, relative canopy leaf area). It has been elaborated to be applicable
for evergreen species by Grote (2007) who described leaf development for each leaf
age class separately. The second impact is a description of synthase turnover:

f .S / D S0 C Œg.SF / C h.SD /t (12.21)

where S0 is the previous (or initial) state of the seasonality function, g(SF ) is a
function that describes the rate of protein synthesis in dependence on past light and
temperature conditions and phenological state of the leaves, and h(SD ) is a function
that describes the rate of protein degradation (for details see Grote et al. 2010). In
contrast to the previous approaches, this model introduces some mechanistic cause-
effect relationships by considering the increase of enzyme activity as dependent on
absorbed radiation and its decay as a function of temperature.
Another approach has been presented with the Model of Emissions of Gases
and Aerosols from Nature (MEGAN) (Guenther et al. 2006). In this model, age
effects are described by separating the foliage among new, young, and recently
matured leaves. The seasonality aspect was described by adjustment of ES (Eq. 12.1)
independent of phenology in dependence on the temperature of the previous days:

f .S / D cS8 exp ŒcS9 .T24  TREF / exp ŒcS9 .T240  TREF / (12.22)

where cS8 and cS9 are empirical parameters, TREF is a reference temperature
(297 K), and T24 and T240 are average temperatures for the previous 24 and 240 h,
respectively.
In MEGAN, the overall response represents a sine function while, the SIM
approach follows the general pattern of an exponential response, which generally
provides a better fit to data. We present some of the approaches that have been used
for seasonal adjustment of EMAX in Fig. 12.4. The shapes of the seasonal responses
and their maxima near day of year of 200 are generally conserved. However, the
slopes of the responses for the ascending and descending trajectories on either side
of the maxima differ, and this is where model-dependent differences are likely to be
greatest.
Overall, we note that modelling seasonality remains a challenging task. As accli-
mation and age effects cannot be deconvoluted, it is important to be aware that the
seasonality and age models may partly include acclimation effects. This understand-
ing is relevant especially when trying to incorporate various acclimation, stress, and
seasonal controls in multivariate models (Eq. 12.1) to avoid “double-counting” of
various factors, thereby over-parameterizing the model (Niinemets et al. 2010a).
334 R. Grote et al.

1.2
MEGAN
SIM
1.0

Relative emission rate

STAUDT

0.8

0.6

0.4

0.2

0.0
0 60 120 180 240 300 360
Day of the year

Fig. 12.4 Seasonal adjustment of maximum emission rate in broad-leaved sclerophyll Quercus
ilex according to an empirical fit to data from Staudt et al. (2000), and the emission capacity
predicted according to weather-dependent MEGAN (Guenther et al. 2006) and SIM (Lehning et al.
2001) approaches using the weather conditions at Montpellier, France for 2006 growing season
(Modified from Grote et al. 2010). The emission rates were normalized to the highest observed
value

12.3.2 Acclimation to Variations in Light Environment

Several studies have demonstrated that both isoprene emission capacity (Harley
et al. 1996, 1997; Geron et al. 1997; Hanson and Sharkey 2001; Funk et al. 2006;
Niinemets et al. 2010a, b) and monoterpene emission capacity in “non-storage”
species (Lenz et al. 1997; Niinemets et al. 2002a; Staudt et al. 2003) increases
with increasing long-term light availability. In particular, extensive within-canopy
variation in isoprene emission rate of 3-27-fold has been recently demonstrated in
broad-leaved deciduous trees (Fig. 12.5). Depending on within-canopy plasticity in
isoprene emission potential, model estimates of whole canopy isoprene emissions
using a constant emission factor are biased by 8 to C68 % (Niinemets et al.
2010b). Guenther et al. (1999) linked such within-canopy variations at the level
of coefficient cL3 of the light response function of Eq. 12.11. Thus, the coefficient
cL3 essentially functions as a scaling factor. As no long-term light measurements
were available, cL3 was linked to cumulative leaf area index (Lcum ) as (Guenther
et al. 1999):

cL3 D 1:42 exp .0:3Lcum / : (12.23)

However, foliage acclimates to long-term quantum flux density, Qint , rather than to
Lcum , and Qint corresponding to a given value of Lcum may vary in dependence on
foliage angular distribution and spatial aggregation (Cescatti and Niinemets 2004).
Despite species-specific variations in the within-canopy variability of emission
capacity, Niinemets et al. (2010b) demonstrated that when all data across the species
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 335

Fig. 12.5 Variation of a

isoprene emission rate at
standard conditions of leaf
temperature of 25 ı C and
incident quantum flux density
of 1,000 mol m2 s1

−2 −1
(emission factor) with
seasonal average integrated Qr
quantum flux density (Qint ) in 30
four temperate deciduous
species (a), and the emission Pt
Sa Sc
factor standardized to the 20
above-canopy (average
seasonal maximum) quantum
flux density of 37.3 mol m2
10
day1 (modified from
Niinemets et al. 2010b). Data
in (b) are fitted by the
so-called canopy function of 0
isoprene emission (f (C),
Eq. 12.24). Representative b
1.2
hemispheric photographs
demonstrating different light f Q int
1.0
environments within the
canopy are also shown 0.8
(arrows denote the Qint values
corresponding to the four 0.6
hemispheric photographs)
0.4
37.3
−2 −1
0.2

0
0 10 20 30 40

−2 −1

were standardized to above-canopy seasonal average Qint (37.3 mol m2 day1 in
their study), the variation decreased (Fig. 12.5) and all data could be fitted by a
single canopy function, f (C):

f .C / D 0:843Log .0:411Qint / : (12.24)

The bias of using Eq. 12.24 in estimating whole canopy isoprene emission flux
relative to the use of species-specific variation patterns was only 11 % to C6 %
(Niinemets et al. 2010b). Thus it would be more accurate to use Eq. 12.24 instead
Eq. 12.23 in future emission models.
The other parameter, susceptible to acclimation is the quantum yield (’ in
Eq. 12.11) used to define the instantaneous light response of immediate emission.
This parameter can vary within the canopy (Fig. 12.2b) due to changes in leaf
336 R. Grote et al.

chlorophyll content (Niinemets 2007). For isoprene and monoterpenes, studies have
suggested an increasing apparent quantum yield with canopy depth (Harley et al.
1996, 1997; Staudt et al. 2003), and the apparent quantum yield has thus been
expressed in dependence on Lcum similar to cL3 (Guenther et al. 1999):

˛app D 0:001 C 0:00085Lcum (12.25)

However, we note that there is an explicit connection between ’app and cL3 as
indicated in Sect. 12.2.3 and in Monson et al. (2012). In fact, Harley et al. (1997)
fit the light response curves of isoprene emission using Eq. 12.10, and observed
only minor within-canopy variation in the true quantum yield. Thus, it remains to
be tested to what extent the true quantum yield for isoprene emission does indeed
vary in plant canopies.

12.3.3 Needs for Future Developments

Representing acclimation processes of foliage emission at the ecosystem scale is

a difficult task since seasonal development of cell-to-leaf level states are not only
directly affected by environmental conditions, but are also indirectly influenced by
phenological, ontogenetic and structural properties of the emitting plants. This is
most obvious for foliage amount which varies during the year due to leaf flushing
and senescence, and these effects are particularly obvious in deciduous species.
Additionally, ontogenetic changes affect isoprenoid emission potentials (maximum
rate under standardized conditions) (e.g., Grinspoon et al. 1991; Kuzma and Fall
1993; Fuentes and Wang 1999). The capacity to emit isoprenoids generally develops
gradually during leaf development and reaches a maximum only after full leaf
expansion; following maximum leaf expansion, and the emission potentials further
gradually decrease with increasing leaf age (Fischbach et al. 2002). With respect
to evergreen species, it is thus important that functional activity continuously
decreases with increasing leaf age (Niinemets et al. 2006, 2013; Grote 2007).
Finally, canopy structure determines microclimatic conditions that affect short- and
long-term impacts on emission processes throughout the canopy (Keenan et al.
2011). All of these vegetation processes develop dynamically and simultaneously
in response to changes in the seasonal environment. It is difficult to disentangle
the direct and indirect seasonal influences on emission potential and to define
species-specific differences in acclimation capacity, principally due to the lack
of empirical information. For example, surprisingly little information is available
on emission potentials in older leaves. Other physiological developments such as
seasonal dynamics in isoprenoid storage pools, which are not yet considered in any
model (Schurgers et al. 2009) add to these uncertainties. Finally, emissions related
specifically to bud, flower, and fruit development are not addressed in models,
although modified emission patterns – qualitatively and quantitatively – have been
reported for the period of bud burst (e.g., Kuhn et al. 2004).
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 337

12.4 Incorporating Stress in Models of Constitutive Emission

Stress can have several effects on volatile emissions. First, in constitutively emitting
species, stress may modify the emission capacity and/or the shape of emission
responses to environmental drivers. Second, stress can lead to induction of volatile
emissions in both emitting and (otherwise) non-emitting species. As natural veg-
etation is often under stress, even suffering frequently from co-occurring and
sequential stress episodes (Loreto and Schnitzler 2010; Niinemets 2010a, b), our
ability to predict stress responses on volatile emissions is urgently needed for
reliable prediction of emission time series. In this section, we analyse how stress
effects on constitutive emissions can be incorporated in emission models focusing
on the influences of altered transfer conductances and biochemical modifications as
exemplified by drought responses. For pollutant effects on constitutive emissions
we refer to Calfapietra et al. (2013) in this volume.

12.4.1 Impacts on Conductances

12.4.1.1 Stomatal Controls

Constitutive emissions are controlled by temperature and the diffusive resistances

between storage pools and the atmosphere. Several past studies have focused on
stomata as the primary resistance to emission from internal storage pools. In the
early studies of foliage isoprene emission, it was recognized that the steady-state
isoprene emission rate is independent of stomatal conductance (Gs ) (Monson and
Fall 1989; Fall and Monson 1992). Fall and Monson (1992) hypothesized that
steady-state reductions in Gs were compensated by increases in
p, the difference
in isoprene partial pressure between the intercellular air spaces of the leaf (pi ) and
the ambient atmosphere (pa ). Thus, E D Gs (p/P), where P is the air pressure. The
theory underlying this relation and its application to a range of emitted BVOCs
requires that for compounds which have relatively high Henry’s law constants
(gas/liquid phase partition coefficients), perturbations in Gs should result in rapid
(within seconds) establishment of a new diffusion steady state (Niinemets and
Reichstein 2003). This would not be true for BVOCs with lower Henry’s law
coefficients (e.g., oxygenated isoprenoids, organic acids or methanol). Niinemets
and Reichstein (2003) formalized the theory on these relations by stating:

.pi  pa / .H Cw  pi /
E D Gs D GL (12.26)
P P

where H is the Henry’s law constant for the particular BVOC (Pa m3 mol1 ), Cw is
its concentration in the liquid (water) phase of the cell or cell wall (mol m3 ), and GL
is the gas-phase equivalent of liquid phase conductance from the site of compound
synthesis to the outer surface of cell wall. Implicit in Eq. 12.26 is that compounds
338 R. Grote et al.

with low H support a lower vapour pressure for given liquid phase concentration,
and accordingly, the diffusion gradient, p, increases slowly such that changes
in stomatal conductance can transiently limit volatile emissions (Niinemets and
Reichstein 2003; Harley 2013).

12.4.1.2 Breakage of Storage Structures

Enhancements of emissions of stored BVOCs occur when leaf tissue is wounded

and broken epidermis and cuticle strongly decrease diffusive resistances. These
effects are particularly relevant in characterizing the impact of logging operations in
forests where terpene-filled tissue is destroyed (e.g., Strömvall and Petersson 1991).
Similarly, insect attacks can open plant storages of volatile compounds that often
act as a defence and serve to poison or otherwise deter attackers (Loreto et al. 2000;
Trowbridge and Stoy 2013). We note that past relationships of terpene content vs.
emission rate as shown for some conifers (Lerdau et al. 1994, 1995) may reflect the
“rough handling problem”, i.e., exposure of internal storage structures to ambient
air during measurements.
To date, the effects of rapid changes in diffusion conductance from the site of
storage to ambient air have not been considered in emission models. However,
BVOC pools have previously been quantified (e.g., Llusià and Peñuelas 1998; Llusià
et al. 2010) and their release can be simulated according to Eqs. 12.2 or 12.3 with
the additional assumption of a limited (and decreasing) storage pool size (Schurgers
et al. 2009). Nevertheless, the models likely need to be more complex than just first
order decay functions, because the initial rapid increase in emissions is followed
by time-dependent reduction of the emission rates as the wound becomes sealed,
e.g., as the result of oxidation and polymerization of oleoresin components (Loreto
et al. 2000).

12.4.2 Impacts on Biochemistry

The impact of drought has been studied in several investigations as time-integrated,

long-term influence on isoprene emission (e.g., Fang et al. 1996; Brüggemann and
Schnitzler 2002; Pegoraro et al. 2004; Brilli et al. 2007). However, until recently,
drought has not been considered as a modifier in BVOC emission models. Drought
can influence the emissions in three ways. First, reductions in leaf evaporative
cooling due to constrained leaf transpiration rates, leading to concomitant increases
in TL . Second, decreases in stomatal conductance result in reduced Ci . Finally, there
can be direct effects of drought on metabolic processes.
The first effect can be accommodated in the models by considering the deviations
between TL and air temperature. The second influence can be incorporated through
Eqs. 12.15, 12.6 and 12.17 when properly parameterized to consider reduced CO2
growth regime, especially considering the reduction of emissions below a critical Ci .
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 339

Modelling the effects of metabolic modifications is most complex. Drought tends to

trigger a cascade of metabolic feedbacks that function to balance metabolism with
growth potential. Grote et al. (2009) took advantage of previous studies of changes
in the concentrations of certain photosynthetic metabolites to represent drought
effects on monoterpene emissions through the availability of BVOC precursors. One
premise of this approach is that a tight coupling exists between leaf carbon balance,
as influenced by leaf photosynthesis rate, and isoprenoid emission. However, this
assumption neglects the shift in resources between different biochemical pathways
under stress. Within the MEGAN model, Guenther et al. (2006) introduced a
drought scaling factor as a linear relation between relative water availability and
E as an additional multiplier in Eq. 12.1. This function was defined as:
8
ˆ
ˆ 1; if   1
<
.  W /
f .W / D ; if W <  < 1 (12.27)
ˆ 1
:̂
0; if   W

where  is the extractable water content (m3 m3 ),  w is the soil water content
at leaf wilting point, i.e., the soil water content that cannot be extracted by plant
roots,  1 is an empirically-determined soil water limit that can be expressed as
 1 D  w C  1 .  1 is commonly set as 0.06 following Pegoraro et al. (2004).
One of the difficulties with using this type of model is the determination of  w as
well as  1 . Guenther et al. (2006) used the wilting point database of Chen and
Dudhia (2001) for global emission estimation. However, there are no studies to date
that have established the wilting point as a conserved and relevant determinant of
drought stress on photosynthesis or BVOC emission.
The greatest barrier to progressing in our ability to model drought stress effects
on BVOC emission is our incomplete understanding of the metabolic connections
among drought, expression of BVOC synthase activities, availability of BVOC
substrates, and drought-induced changes in the sensitivities of BVOC formation to
light, temperature and intercellular CO2 concentration. Future studies should focus
on these connections, which may allow us to integrate drought-stress models more
effectively into BVOC models.

12.5 Simulation of Induced Emissions

12.5.1 General Patterns

Consistent with the theory and evidence that BVOC emissions serve primarily
as a protection against abiotic stress and for communication among ecological
tropic levels (Holopainen 2004; Sharkey et al. 2008). BVOC emissions can be
induced by practically any stress factor in species emitting and non-emitting
volatiles constitutively (Heiden et al. 2003). The emission of stress volatiles reflects
340 R. Grote et al.

elicitation of defence pathways, side-products of intermediates of which are volatile,

and synthesis of volatile products with known or yet unknown functions in direct and
indirect defence (Pare and Tumlinson 1999, Kessler and Baldwin 2001; Peñuelas
and Llusià 2003; Owen and Peñuelas 2005, 2006; Niinemets 2010a, b). Induction
of volatile emissions has been demonstrated in response to both biotic stresses such
as insect herbivory (e.g., Priemé et al. 2000; Miller et al. 2005; Dicke et al. 2009;
Copolovici et al. 2011; Blande et al. 2009), and fungal pathogens (Steindel et al.
2005; Toome et al. 2010) and abiotic stress such as UV radiation (e.g., Blande
et al. 2009), ozone (e.g., Beauchamp et al. 2005; Blande et al. 2007), heat and frost
(Loreto et al. 2006; Copolovici et al. 2012), flooding (Copolovici and Niinemets
2010; Kreuzwieser and Rennenberg 2013), and mechanical wounding (Fall et al.
1999; Banchio et al. 2005; Loreto et al. 2006).
Emissions of early stress volatiles during and immediately after stress reflect
activation of signalling at the level of membranes and cell walls and are associated
with the release of methanol (Beauchamp et al. 2005; Loreto et al. 2006; von Dahl
et al. 2006; Copolovici and Niinemets 2010) and green leaf volatiles (various C6
aldehydes) (Priemé et al. 2000; Loreto et al. 2006; Copolovici and Niinemets 2010;
Copolovici et al. 2011, 2012; Blande et al. 2007, 2009; Kirstine and Galbally 2004;
Loreto et al. 2006; Davison et al. 2008; Brilli et al. 2012). These emissions are
followed by activation of gene expression and emissions of specific volatile iso-
prenoids from stressed foliage (Dicke 1994; Paré and Tumlinson 1997; Beauchamp
et al. 2005; Toome et al. 2010; Copolovici et al. 2011; Blande et al. 2007, 2009).
Furthermore, release of volatiles and synthesis of non-volatile phytohormones in
stressed leaves can elicit systemic response in neighboring non-stressed leaves of
the same plant and in neighboring different plants, resulting in volatile emissions of
apparently healthy leaves (Dicke 1994; Röse et al. 1996; Paré and Tumlinson 1998;
Staudt and Lhoutellier 2007; Holopainen et al. 2013; Trowbridge and Stoy 2013).
Characteristic stress-induced volatile isoprenoids are monoterpenes linalool and
ocimenes, homoterpenes DMNT and TMTT and various sesquiterpenes (Loivamäki
et al. 2004; Herde et al. 2008; Dicke et al. 2009; Toome et al. 2010), and
thus, the composition of elicited isoprenoids typically differs from the volatiles
released in non-stressed conditions (Loreto and Schnitzler 2010; Niinemets et al.
2010c; Schnitzler et al. 2010). As noted above, in constitutively emitting species,
biotic or abiotic stress may result in suppression of constitutive emission rates
(Anderson et al. 2000; Copolovici and Niinemets 2010; Toome et al. 2010), but
not always (Calfapietra et al. 2007, 2008; Copolovici and Niinemets 2010). Yet, in
constitutively non-emitting species, volatile emissions generally increase from low
background level by several orders of magnitude even above the level observed in
constitutively emitting species (Niinemets et al. 2010c for a review). For instance,
temperate deciduous broad-leaved birch (Betula) species have been observed to
emit mono- and sesquiterpenes at a low level of only 0.1–0.4 g g1 h1 in
some studies and during certain periods during the growing season (Fig. 12.6,
König et al. 1995; Hakola et al. 1998, 2001). However, under stress conditions,
they have been found to be relatively strong emitters of monoterpenes linalool and
ocimenes, and sesquiterpenes, with standardized emission rates (leaf temperature of
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 341

10

−1 −1
6

Induced
4
Constitutive
2

Fig. 12.6 Variation in standardized monoterpene emission factor (leaf temperature of 30 ı C,

incident quantum flux density of 1,000 mol m2 s1 ) in temperate deciduous birch (Betula)
species (Data from König et al. 1995; Steinbrecher et al. 1997; Hakola et al. 1998; Hakola
et al. 2001; Owen et al. 2003). Sustained emissions under non-stressed conditions are defined as
constitutive emissions, while emissions elicited under certain stress periods are defined as induced
emissions

30 ı C and incident quantum flux density of 1,000 mol m2 s1 ) between 1.5 and
8 g g1 h1 (Fig. 12.6, König et al. 1995; Hakola et al. 1998, 2001; Steinbrecher
et al. 1999; Owen et al. 2003). Analogously, in a constitutive-emitter Mediterranean
evergreen conifer Pinus pinea, total emissions from stressed plants are several-fold
greater than the constitutive emissions from non-stressed plants (Staudt et al. 1997,
2000; Niinemets et al. 2002b, c).

12.5.2 Modelling Induced Emissions

Induction of BVOC emissions can reflect activation of enzymes that are already
present or increased expression of the genes that encode various BVOC synthases.
Given the growing evidence that a considerable fraction of emission responses are
related to stress induction, models that describe these processes are beginning to
emerge. Iriti and Faoro (2009) have suggested differentiating between primary and
secondary metabolic pathways in the induction process with concomitant modifica-
tions in carbon fluxes among the pathways. The mechanism of volatile induction is
complex, starting with signal perception that triggers the cascade of events leading
ultimately to activation of transcription regulators and onset of expression of volatile
synthases (Bolwell et al. 2002; Maffei et al. 2007; Mithöfer and Boland 2008; Loreto
and Schnitzler 2010; Niinemets 2010a; Arimura et al. 2011). The mechanisms
of signal perception and elicitation of gene expression can differ for different
stresses, but there is evidence of a uniform stress response elicitation pathway for
both biotic and abiotic stresses at the level of oxidative signalling (Bostock 2005;
Fujita et al. 2006). Often, there is also a cross-talk between ethylene-, salicylate-
and jasmonate-dependent stress response pathways (Thaler et al. 2002; Traw and
342 R. Grote et al.

Bergelson 2003; Bostock 2005; Fujita et al. 2006; Mithöfer and Boland 2008). Thus,
general stress response models can in principle be constructed (Niinemets 2010a).
From an experimental perspective, there is increasingly more evidence that stress
severity and plant volatile emission response are quantitatively related, including
positive correlations between the severity of ozone (Beauchamp et al. 2005), heat
(Karl et al. 2008; Copolovici et al. 2012) and insect herbivory (Copolovici et al.
2011) stresses. Scaling of volatile emission response with the stress severity has
been used in predicting methyl salicylate emissions from a walnut (Juglans califor-
nica  Juglans regia) agroforest on the basis of average temperature preceding the
measurements (Karl et al. 2008). While such empirical models based on average
level of environmental drivers can be useful once the emissions have been triggered,
emissions typically are not induced until a certain stress threshold has been exceeded
(Beauchamp et al. 2005; Copolovici et al. 2012), except perhaps for wounding and
insect herbivory that essentially always trigger emissions. Thus, the key issue in
predicting stress induction of volatiles is to determine when a given environmental
driver is sensed as a stress by the plant. The stress thresholds depend on a variety of
factors including plant tolerance to given type of stress and past stress history such
as stress priming (Conrath et al. 2006; Heil and Kost 2006; Heil and Silva Bueno
2007; Niinemets 2010a, b). Thus, a stress of given severity may or may not result in
inductions of volatile emissions.
The second difficulty of simple empirical models is what happens after stress.
When the stress is relieved, for how long do the triggered emissions continue?
There is evidence that after the stress relief, the induced emissions may reach to
a background level in a few days (Copolovici et al. 2011). The emissions may
also decrease during the stress as plant acclimates to the stress (Copolovici and
Niinemets 2010). However, there is also evidence of sustained emissions once
elicited (Staudt et al. 1997, 2000; Hakola et al. 2001; Niinemets et al. 2002b;
Copolovici and Niinemets 2010).
Stress signalling models are currently being intensively developed (Vu and
Vohradsky 2007; Yip et al. 2010; Muraro et al. 2012), but due to limited knowledge
of signal transduction and transcription regulators, completely mechanistic models
cannot yet be derived. We suggest that for the time being, the dynamic controls on
induced BVOC emissions can be simulated based on the theory of recursive action
of regulators on the target gene(s) over time (Vu and Vohradsky 2007; Yip et al.
2010). Thus, the target gene activity change over time, dz/dt, is expressed as (Vu
and Vohradsky 2007; Yip et al. 2010):
dz vmax
D !  kz.t/; (12.28)
dt jP
Dn
1 C exp  wj yj C c
j D1

where vmax is the maximum rate of expression, k is the rate constant of degradation,
z(t) is the gene product amount at time t, n is the number of gene regulators
considered, wj is the weighting factor for a given control function yj , and b
is the delay factor describing the lag in the transcription initiation. Thus, vmax
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 343

Fig. 12.7 Illustration of Larvae Larvae

elicitation of monoterpene added removed
emissions in temperate
deciduous tree Alnus 0.5
glutinosa by the common a
white wave (Cabera pusaria) 0.4
larvae (a), and the
relationships between the 0.3

2 −1
average rate of leaf
consumption and 0.2
monoterpene emission rate
(b) and the rate of 0.1 control
monoterpene synthase
formation (c) (Data from 0
Copolovici et al. 2011). In the 0 1 2 3 4 5 6
experiment, the plants of A.
glutinosa were subject to
0.5
different levels of herbivory b
by using either 0 (control), 2,
0.4
4 or 8 C. pusaria larvae per
plant. The measurements
0.3 r2
were conducted at 28 ı C. The
data in (a) were simulated by
0.2
a model based on dynamic
transcriptional control
0.1
(Eq. 12.28), and the rate of
control
monoterpene synthase
0
formation was found by
fitting the data in (a) by the 6
model. In calculating the c
−1

protein formation rate, a

specific activity of
monoterpene synthase of 4 r2
94 nmol g1 s1 at 28 ı C
was used (Niinemets et al.
2002c), and it was further 2
assumed that the induced
monoterpene synthases
operate in substrate-saturated 0
conditions 0 2 4 6 8

2 −1

and the denumerator determine the onset of gene expression, while kz and wj yi
functions determine the silencing of the response. This model assumes that the
overall regulatory effect on a given gene can be expressed as the combination of
all regulators (Vu and Vohradsky 2007; Yip et al. 2010). Highly plastic non-linear
transcription control effects can be simulated using various linear or non-linear yi
functions, and it has been demonstrated that Eq. 12.28 provides excellent fits to
complex gene expression profiles (Vu and Vohradsky 2007).
Equation 12.28 was applied here to the induction of monoterpene emissions in
the temperate deciduous tree black alder (Alnus glutinosa) (Fig. 12.7a, Copolovici
344 R. Grote et al.

et al. 2011). In this study, different levels of herbivory were achieved by using
either 0 (control), 2, 4 or 8 larvae of the common white wave (Cabera pusaria)
on each plant (Copolovici et al. 2011). The rate of leaf biomass consumption scaled
positively with the number of feeding larvae, and the rate of induced monoterpene
emission was quantitatively related to the rate of foliage consumption (Fig. 12.7a,
Copolovici et al. 2011). In the model fit, only one transcriptional control was
assumed and the control function was described by a fifth order polynomial.
The model applied here provides excellent fits to the data (Fig. 12.7b), and
allows for the estimation of kinetic dynamics in transcription, maximum rates of
induced monoterpene synthase formation and rates of monoterpene synthase decay
under different herbivory treatments. The maximum rate of monoterpene synthase
formation was quantitatively associated with the rate of herbivory (Fig. 12.7c). To
our knowledge, this is the first evidence demonstrating that stress signal strength
can be quantitatively simulated to project target protein synthesis rate. On the
other hand, we also observed differences in the transcription regulation function
in different treatments, with the emissions being both elicited and declining earlier
in the treatment with two than in the treatment with eight larvae (Fig. 12.7a).
Such differences cannot be currently explained, and apart from differences in
plant transcription regulation, might reflect differences in the feeding behavior
of herbivores in different treatments. Overall, this exercise provides encouraging
evidence that models based on dynamic transcription control can be used to simulate
induced emissions, and we suggest that simple dynamic regulatory models such as
Eq. 12.28 together with quantitative relationships between the severity of stress and
maximum plant response have large potential to simulate stress-driven emissions in
larger-scale models.

12.6 Conclusions

Considering the different environmental impacts that affect BVOC emission, it

has become increasingly apparent that integrated descriptions of processes are
beginning to emerge. Such integrated models will permit us to begin examining
higher-order interactions between environmental change and ecosystem BVOC
emissions, including the feedbacks that control regional- to global-level dynamics
in atmospheric chemistry and in the production and lifetime of radiatively-important
trace gases such as O3 and CH4 . In this chapter, we have concentrated on
the leaf-scale modelling as the most significant breakthroughs in recent BVOC
modelling have been made at this scale. There is now increasing recognition that
the mechanistic emphasis that has been in focus at the leaf scale needs to be
expanded to consider processes at greater spatial scales and longer temporal scales
(Guenther 2013 in this volume). Consideration of the latter, takes us into the need
to discover ways of simulating the interactions between environmental cues and
gene expression. Simulation of these larger and longer-term processes will allow
us to begin tackling some of the regional and global dynamics in air chemistry.
12 Leaf-Level Models of Constitutive and Stress-Driven Volatile Organic. . . 345

These controls are increasingly recognized as being central components in Earth

system models (Ashworth et al. 2013; Kulmala et al. 2013), and we argue that more
biological realism needs to be incorporated in these models in near future.

Acknowledgements The work of ÜN on volatile isoprenoid emission has been sponsored by the
Estonian Research Council (Plant stress in changing climates), the Estonian Science Foundation
(grant 9253), the European Science Foundation (Eurocores project A-BIO-VOC), the European
Commission through European Regional Fund (the Center of Excellence in Environmental
Adaptation) and European Research Council (advanced grant 322603, SIP-VOLC).

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Chapter 13
Scaling BVOC Emissions from Leaf to Canopy
and Landscape: How Different Are Predictions
Based on Contrasting Emission Algorithms?

Ülo Niinemets, Paolo Ciccioli, Steffen M. Noe, and Markus Reichstein

Abstract A variety of leaf-level models has been embedded in a canopy model

and used to predict monoterpene emissions from canopies and landscapes, but there
is no objective basis of choice between different models. Here we analysed the
capacity of four leaf-level models and their variations, yielding altogether eight
models, for predicting diurnal and seasonal variations in canopy monoterpene
emissions. The main models tested were Guenther et al. model with fixed light
and temperature dependencies or with optimally adjusted dependencies, two models
linking emissions to foliage photosynthetic rate, one to electron transport rate (ETR
model) and the other to gross assimilation rate (C-ratio model), and a dynamic
model considering non-specific monoterpene storage in leaves. Once parameterized
in a consistent manner, all models showed similarly high performance, assessed
by explained variance, modelling efficiency and average model deviations for
homogeneous canopies. Simulations suggested potentially stronger deviations for
landscapes with fragmented vegetation. This analysis indicates that the choice
among the models cannot be based on model validation statistics alone, but depends
on whether only BVOC emissions need to be simulated (Guenther et al. model)
or both photosynthesis and BVOC fluxes are needed (ETR or C-ratio model) or
whether one needs data on night atmospheric reactivity (dynamic model).

Ü. Niinemets () • S.M. Noe

Department of Plant Physiology, Institute of Agricultural and Environmental Sciences,
Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, Estonia
e-mail: [email protected]
P. Ciccioli
Istituto di Metodologie Chimiche, Consiglio Nazionale delle Ricerche (CNR),
Area della Ricerca di Roma 1, 00016 Monterotondo Scalo, Italy
M. Reichstein
Max Planck Institute for Biogeochemistry, P.O. Box 100164, 07701 Jena, Germany

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 357
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 13,
© Springer ScienceCBusiness Media Dordrecht 2013
358 Ü. Niinemets et al.

13.1 Introduction

Biogenic volatile organic compound (BVOC) emissions play a primary role in

tropospheric ozone formation (Chameides et al. 1988; Simpson 1995). Therefore,
for mesoscale air pollution modelling it is crucial to have reliable estimates of
ecosystem BVOC fluxes (e.g., Simpson 1995; Fuentes et al. 2000). BVOC further
participate in the formation of secondary organic aerosols and cloud condensation
nuclei, thereby playing a major role in large-scale Earth processes (Huff Hartz
et al. 2005; Engelhart et al. 2008; Chen and Hopke 2009; Hallquist et al. 2009;
Guenther et al. 2012; Kulmala et al. 2013 in this volume), further emphasizing
the importance of accurate estimations of BVOC fluxes. The largest part of BVOC
emissions originates from plant leaves (Geron et al. 1994; Simpson 1995; Guenther
et al. 2006), and accordingly, the most significant improvement of estimates of large-
scale emissions may be achieved through an advancement of leaf- and canopy-scale
emission models.
For one of the most volatile plant compounds, isoprene, the synthesis of leaf-
level data has led to development of an empirical model that uses a hyperbolic
light-dependence and an Arrhenius type temperature response to describe the
environmental effects on emission rates (Guenther et al. 1991, 1993; Monson et al.
2012). This model has also been successfully employed to simulate light- and
temperature effects on monoterpene emission in species that lack specific terpene
storage structures in the foliage such as the Mediterranean evergreen Quercus
species (e.g., Bertin et al. 1997; Ciccioli et al. 1997) and the broad-leaved deciduous
tree Fagus sylvatica (Dindorf et al. 2006; Holzke et al. 2006). However, in the case
of less volatile monoterpenes, non-specific storage effects have been demonstrated
that can potentially introduce time-lags between the synthesis and emissions of
these compounds (Niinemets and Reichstein 2002; Noe et al. 2006, 2008, 2010),
potentially altering the daily emission dynamics. While these effects have been
characterized at leaf scale, their importance at canopy scale has not been assessed.
Although models with fixed response shapes can realistically describe the light
and temperature effects on the emission rates in non-stressed conditions and in
fully-developed leaves, they cannot adequately predict the emission rates in stress
conditions, and they also have limited potential to account for potential changes
in the response curves that may occur as the result of leaf acclimation to varying
environmental conditions (Berry and Björkman 1980; Mulkey et al. 1991). In
particular, drought stress regularly occurs in Mediterranean environments, leading
to a gradual decrease in maximal stomatal conductances to water vapour with
advancement of soil water limitations, and typically also to mid-day stomatal
closure (Tenhunen et al. 1987; Manes et al. 1997b; Peñuelas and Llusià 1999).
Decreases in stomatal conductance not only result in decreased net assimilation
rates, but also in declines in the emission rates of monoterpenes (Moncrieff et al.
1997; Ciccioli et al. 1999; Hansen and Seufert 1999; Peñuelas and Llusià 1999;
Niinemets et al. 2002b). To parameterize such stress effects, a series of empirical
modifiers has been recently included in Guenther et al. algorithms (Guenther et al.
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 359

2006, 2012; Monson et al. 2012; Grote et al. 2013 in this volume). Stress-related
changes in the emission rates might be more adequately described by physiological
models resting on the correlations between the emission rate and the rate of
synthesis of Calvin cycle intermediates (Martin et al. 2000; Zimmer et al. 2000)
or between the emission rate and the supply of reductive and energetic equivalents
by photosynthetic electron transport (Niinemets et al. 1999, 2002a, c). However, the
ultimate process-level descriptions in these models are limited by the circumstance
that the physiological controls on isoprene and monoterpene emissions have still not
been fully resolved.
There are also significant seasonal changes in the isoprene (Monson et al. 1994,
2012; Schnitzler et al. 1997; Lehning et al. 2001) and terpene emission capacities
(Hakola et al. 1998; Llusià and Peñuelas 2000; Staudt et al. 2000; Sabillón and
Cremades 2001; Keenan et al. 2009) associated with developmental changes in
the content of enzymes controlling the pathway flux. Seasonality effects have been
included in recent emission models (e.g., Guenther et al. 2006; Keenan et al. 2009),
although the parameterizations largely differ (Monson et al. 2012; Grote et al. 2013
in this volume). For instance, in the Guenther et al. (2006) model, seasonality is
directly related to leaf area development, and aging, but also results from longer-
term seasonal changes in environmental drivers. Although the approach used is
plausible, the model equations postulated were parameterized on the basis of a
limited number of case studies.
Here we compare the potentials of four different leaf-level monoterpene emission
approaches to predict monoterpene fluxes at the canopy and landscape levels.
In addition to the widely used Guenther et al. (1993) algorithm, two additional
approaches linking the monoterpene emission rate either to photosynthetic electron
transport (Niinemets et al. 1999, 2002a, c) or to the fraction of assimilated carbon
lost as monoterpenes (Martin et al. 2000, current study) were included. Finally,
a dynamic model considering non-specific monoterpene storage (Niinemets and
Reichstein 2002) was used. At canopy scale, all models were evaluated against
relaxed eddy accumulation flux measurements conducted in a Mediterranean
evergreen Quercus ilex L. stand, while landscape-level estimates were compared
in a hemiboreal mixed forest.

13.2 Leaf-Level Monoterpene Emission Models: Emission

Algorithms and Scaling

A variety of emission algorithms has been developed to simulate light- and

temperature-dependent volatile isoprenoid emissions from the foliage of emitting
plants. The model equations have been reviewed in detail recently (Monson et al.
2012; Grote et al. 2013 in this volume), and here the models are only briefly
described in order to highlight the characteristic features (Table 13.1) and explain
the way they were parameterized in the intercomparison exercise. We consider
360 Ü. Niinemets et al.

separately the steady-state models that assume instantaneous response of emis-

sions to changes in incident quantum flux density (Q) and leaf temperature (T),
and dynamic models that consider potential time-lags between the synthesis and
emission of monoterpenes. This may be relevant given that due to limited volatility
of monoterpenes, they are non-specifically stored in leaf liquid and lipid phases even
in species not having specialized storage structures (Niinemets and Reichstein 2002;
Niinemets et al. 2004; Noe et al. 2010).

13.2.1 Emission Algorithms

We analysed the performance of three different steady-state emission algorithms

and one dynamic emission model for estimating the monoterpene emission rate
E (Table 13.1). The selected algorithms cover a spectrum of models including
models with fixed response shapes directly simulating emissions, models linking
emissions to foliage photosynthetic traits and thus, indirectly simulating emissions
and models that consider time-lags between compound synthesis and release (for
detailed analysis of emission models see Niinemets et al. 2010c; Monson et al. 2012;
Grote et al. 2013 in this volume).

13.2.1.1 Guenther et al. Model

In the Guenther et al. G93 algorithm (Guenther et al. 1993), an estimate of E

in standardized conditions (ES , T D 30 ı C, Q D 1,000 mol m2 s1 ) is scaled
to combinations of leaf temperature and light using predetermined shapes of
temperature (CT ) and light (CL ) response curves (Table 13.1). To describe seasonal
changes in ES , several approaches have been offered (Guenther et al. 2006; Keenan
et al. 2009; Niinemets et al. 2010a; Monson et al. 2012; Grote et al. 2013 in this
volume). We have tested different symmetric and asymmetric functions and finally
we used the following empirical function to describe changes in ES as a function of
day of the year (D):
(
ES;max e .aCb=DCc ln D/ ; if D < D200
ES D 
.d D/2 ; (13.1)
ES;max e 2f 2 ; if D  D200

where a, b, c, d and f are empirical best fit parameters and ES,max is the maximum
value of the basal emission measured during a year. This model that uses two
different equations to describe the emission factor before reaching the maximum
and beyond the maximum is extremely plastic and allows both for simulation of
symmetric and asymmetric seasonality responses.
Table 13.1 Characteristics of key emission models for simulation of light-dependent monoterpene emission rate (E)
Modela Key equation Light dependence Temperature dependence


˛cL1 Q cT1 .T  Ts / cT2 .T  TM /
Guenther E D ES CT CL , where ES is the monoterpene emission rate CL D p , where CT D exp 1 C exp ,
et al. at T D 30 ı C and Q D 1,000 mol m2 s1 , and CL is 1 C ˛ 2 Q2 RTs T RTs T
1 -1
(G93) the light and CT the temperature response function ’ and cL1 are parameters where cT1 (J mol ), cT2 (J mol ) and TM (K) are
model empirical coefficients, Ts is the temperature in
standard conditions (303 K), and R is the gas
constant (J mol1 K1 )
.Ci    /
ETR model E D " JCO2 CO2 , Results from light ©.T / D "Tref e aT , where a is an empirical constant,
12 .4Ci C8  / C 2 .Ci    / NADPH dependence of JCO2 CO2 and ©Tref is the © value estimated at a reference
where © is the fraction of electrons in monoterpene
temperature Tref (30 ı C)
synthesis, JCO2 CO2 is the photosynthetic electron
transport rate, NADPH is the difference of NADPH
requirements for sugar and monoterpene synthesis, Ci is
the intercellular CO2 concentration and  * the CO2
compensation point without dark respiration
C-ratio E D rC AG , where rC is the ratio between monoterpene Combined non-linear empirical equation describing the dependence of rC on both
model emission and gross assimilation rate (AG , Box 13.1) Q and T (Box 13.1, Eq. 13.B2)
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . .

Dynamic E.t / D k1 S1 .t / C k2 S2 .t /, where k1 is the rate constant for Either G93 or ETR model Either G93 or ETR model for monoterpene synthesis
model the faster pool with size S1 (Eqs. 13.3 and 13.5) and k2 for monoterpene rate, temperature dependence of k1 and k2 is
that for the slower pool with size S2 (Eqs. 13.4 and 13.6) synthesis rate predicted by Eq. 13.7
a
G93 – (Guenther et al. 1993), ETR model – (Niinemets et al. 1999, 2002c), C-ratio model (Box 13.1), dynamic model – (Niinemets and Reichstein 2002; Noe
et al. 2006)
361
362 Ü. Niinemets et al.

13.2.1.2 Photosynthetic Electron Transport Model (ETR Model)

A correlation between the whole-chain photosynthetic electron transport rate

(JCO2 CO2 , mol m2 s1 ) and E is employed in the ETR model (Table 13.1,
Niinemets et al. 1999, 2002c; Arneth et al. 2007). The primary assumption of this
model is that the rate of isoprenoid synthesis is directly related to the content of
photosynthetic metabolites and/or NADPH and ATP provided by photosynthetic
electron transport (Loreto and Sharkey 1993). Because volatile isoprenoids are
more reduced molecules than sugars, NADPH and ATP cost per mol C in given
isoprenoid is greater than the cost per C for the formation of sugars in photosynthesis
(Niinemets et al. 1999; Sharkey and Yeh 2001; Niinemets 2004). However, because
the rate of carbon loss through the emission of volatile isoprenoids is generally
much less than the rate of carbon fixation, overall requirement for NADPH and ATP
for isoprenoid synthesis is relatively small compared with photosynthetic carbon
assimilation and photorespiration. Thus, a direct competition for NADPH and ATP
among isoprenoid synthesis pathway and photosynthesis would not explain the
control of isoprenoid synthesis by photosynthetic electron transport rate. However,
NADPH and ATP may exert the control over isoprenoid synthesis pathway if the
effective KM of volatile isoprenoids for NADPH and/or ATP is relatively large
(Rasulov et al. 2009, 2011).
According to the ETR model, a certain fraction of electrons (©) is available for
monoterpene production:

JM C JE
©D (13.2)
JCO2 CO2

where JM is the electron transport rate required to reduce the carbon emitted
as monoterpenes to the carbon reduction state in sugars, and JE is the extra
electron transport rate necessary to reduce the sugars to monoterpenes. The ratio
© characterizes the degree to which photosynthetic electron transport is used for
monoterpene synthesis, and it depends on total activity of monoterpene synthases
(Niinemets et al. 2002c). The conversion from electron to monoterpene units,
mol electrons in monoterpene synthesis (mol monoterpene synthesized)1, is
calculated according to 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose-
5-phosphate (MEP/DOXP) pathway for isoprenoid synthesis (Lichtenthaler 1999).
Considering a composition-weighted NADPH cost for the monoterpenes emitted by
Quercus ilex, a value of 28 mole NADPH per one mole monoterpene emitted is
obtained (Niinemets et al. 2002c). The fraction of electrons used for monoterpene
synthesis can practically be determined at given leaf temperature from each
measurement pairs of JCO2 CO2 and E, and is thus, analogous to the basal emission
rate, ES , in the Guenther et al. algorithm.
In the ETR model, E responds to Q directly through changes in total photo-
synthetic electron transport rate. Temperature dependence is simulated considering
that monoterpene synthesis pathway becomes more competitive for electrons with
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 363

increasing temperature. © at a reference temperature Tref (©Tref ) is scaled to any

other temperature using an exponential relationship (Table 13.1). The seasonality
in © is achieved by modifying the ©Tref parameter according to a function with a
maximum similarly as in the Guenther et al. model (Eq. 13.1) and in the C-Ratio
model (Box 13.1, Eq. 13.B3).

Box 13.1: The C-Ratio Model of Isoprenoid Emission

While linkage of isoprenoid emission rate to leaf physiological activity is
relatively complex and/or requires availability of physiological traits that are
not routinely available in field studies (Monson et al. 2012; Grote et al. 2013
in this volume), we explored the possibility of directly linking isoprenoid
emissions (E) to leaf gross assimilation rate (AG ). There are often correlations
between E and assimilation rate (Monson and Fall 1989; Loreto and Sharkey
1990; Peñuelas and Llusià 1999; Llusià and Peñuelas 2000; Staudt et al. 2001,
2003), indicating that a certain foliage photosynthetic activity is associated
with limited variation in the fraction of carbon going in isoprenoid synthesis.
Given that the assimilation rate can be estimated more easily than either E or
the rate of photosynthetic electron transport rate, a simpler model based on
AG may have a large potential in scaling monoterpene fluxes. In this model
(the C-ratio model), the monoterpene emission rate is calculated as:

E D AG rC ; (13.B1)

where rC is the ratio between monoterpene emission and CO2 gross as-
similation [mol monoterpene emitted (mol CO2 assimilated)1 ]. We note
that correlations between photosynthesis and monoterpene emission rate do
not necessarily indicate that photosynthetic activity determines monoter-
pene emission rate, but rather that there are general correlations between
overall changes in foliage physiological activity and photosynthesis and
isoprenoid emission rate (e.g., Staudt et al. 2003; Sun et al. 2012). Thus,
Eq. 13.B1 provides a means to empirically link monoterpene emissions
to photosynthetic activity. Given that the activity of monoterpene synthase
may increase relatively more with increasing light and temperature than the
activity of enzymes limiting photosynthetic electron transport (Niinemets
et al. 2002a, c), and that monoterpene synthase activity also strongly varies
seasonally (Melle et al. 1996; Fischbach et al. 2002), we expected rC to be
also responsive to these factors. In fact, E was more strongly linked to the
fraction of photosynthetic carbon used for isoprenoid synthesis than to AG in
some studies (Niinemets et al. 2010b; Guidolotti et al. 2011), and this has
been suggested to be indicative of carbon availability control on isoprenoid
emission (Guidolotti et al. 2011).

(continued)
364 Ü. Niinemets et al.

Box 13.1 (continued)

For the Mediterranean sclerophyll Quercus ilex, the C-ratio model was
parameterized using the enclosure data of Ciccioli et al. (2001). These data
demonstrated that rC increased non-linearly with increasing leaf temperature,
T, and incident quantum flux density, Q, (Fig. 13.B1):
 a2
a1 .T 30/ Q
rC D rC;S e ; (13.B2)
1; 000

where rC,S (mmol mol1 ) is rC at standardized conditions

(Q D 1,000 mol m2 s1 , T D 30 ı C), and a1 and a2 are empirical
coefficients.
Data further indicated that rC varied seasonally, likely reflecting changes
in monoterpene synthase activity (Fig. 13.B2). The seasonal variation in rC,S
(Fig. 13.B2) was described by an equation with a maximum rC,S (rmax ) at day
of the year (D) Dmax :
( 2
rmax e kup .DDmax / ; if D < Dmax
rC;S D 2 (13.B3)
rmax e kdown .DDmax / ; if D  Dmax

where kup and kdown determine the rate with which rC,S declines before and
after Dmax . Equations 13.B1, 13.B2 and 13.B3 provide the full set of equations
needed to simulate E for any combinations of Ag , T, Q and D.

13.2.1.3 C-Ratio Model

As an alternative to the ETR model, we also tested a simpler possibility of linking

E directly to the rate of gross carbon assimilation (AG , Box 13.1). The advantage
of the C-ratio model is that it does not require estimation of photosynthetic electron
transport rate, but it still makes a connection to overall changes in leaf physiological
activity (Table 13.1). Use of gross rather than net CO2 assimilation increases
the correlation between assimilation rate and monoterpene emission rate under
conditions of high temperature, when net assimilation rate may decline, but dark
respiration rate (Hüve et al. 2011) and monoterpene emission rate (Loreto et al.
1998) increase. Nevertheless, as with the ETR model, the C-ratio model predicts
that the fraction of carbon going to monoterpene synthesis (monoterpene emission
to AG ratio, rC ) increases with increasing T, but also with Q, albeit weaker than with
T (Fig. 13.B1). Seasonality in rC is expressed similarly as in Guenther et al. and
ETR models (Box 13.1).
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 365

−1

1500
0
−1
40 1000 −2
30
500
20
10 0

Fig. 13.B1 Dependence of the ratio between monoterpene emission and CO2 assimilation (rC ) on
leaf temperature (T) and incident quantum flux density (Q) in Mediterranean sclerophyll Quercus
ilex (data of Ciccioli et al. 2001 measured in saplings grown in 50 L pots). The fitted surface
corresponds to Eq. 13.B2 with best fit coefficients of 0.131 ı C1 for a1 and 0.210 for a2 (r2 D 0.96)
−1

r2 r2
0
0 100 200 300 0 100 200 300

Fig. 13.B2 Seasonal variation of the ratio of monoterpene emission to gross assimilation rate
under standard conditions of T D 30 ı C and Q D 1,000 mol m2 s1 (rC,S ) in current- and
1-year-old leaves of Mediterranean evergreen sclerophyll Quercus ilex. The data were fitted by
Eq. 13.B2 with rmax D 1.92 mmol mol1 , kup D 3.8. 104 , kdown D 1.6. 105 , and Dmax D 200
obtained for current year leaves and rmax D 2.48 mmol mol1 , kup D 1.4. 104 , kdown D 7.1. 105 ,
and Dmax D 149 for 1-year-old leaves (data of Ciccioli et al. 2001 measured in saplings grown in
50 L pots from May 1997 to September 1998)
366 Ü. Niinemets et al.

13.2.1.4 Dynamic Emission Model

To account for non-specific storage of monoterpenes with low volatility in leaf

liquid and lipid phases, Niinemets and Reichstein (2002) and Noe et al. (2006) have
developed a dynamic emission model (Harley 2013 in this volume for a review).
According to experimental data (Niinemets and Reichstein 2002; Noe et al. 2006,
2010), at least two pools, a liquid-phase pool S1 (nmol m2 ) and a lipid-phase pool
S2 (nmol m2 ) with differing time-response (time constants k1 and k2 , s1 ) are
needed to simulate monoterpene emission rate at time t (Niinemets and Reichstein
2002; Noe et al. 2006). The emission rate is calculated as the sum of the turnover
rates of these pools (Table 13.1) with the pool dynamics described as:

dS1 .t/
D I  k1 S1 .t/ (13.3)
dt

dS2 .t/
D .1  / I  k2 S2 .t/: (13.4)
dt
where  is the fraction of synthesized monoterpene going in pool S1 and I the rate
of monoterpene synthesis. The analytical solution of the model is (Niinemets and
Reichstein 2002):
 
I I
S1 .t/ D S1 .t0 /  e k1 t C (13.5)
k1 k1
 
.1  / I .1  / I
S2 .t/ D S2 .t0 /  e k2 t C : (13.6)
k2 k2

The rate of compound synthesis, I, can be simulated by any of the three leaf-level
algorithms (Sects. 13.2.1.1, 13.2.1.2 and 13.2.1.3), but it is important to consider
that the key model parameter determining the emission capacity, ES in Guenther
et al. model, ©TRef in ETR model and rC in C-ratio model, is not numerically the
same as that for the steady-state model (Sect. 13.3.2.1).
Apart from the environmental controls on synthesis, temperature also affects the
kinetic constants k1 and k2 that depend on monoterpene-specific physico-chemical
characteristics (diffusivity in liquid and lipid phases, partition coefficients). The
values of the rate constants k1 and k2 measured at a given absolute leaf temperature
of T1 (K) were estimated at another temperature T2 by the van’t Hoff equation (e.g.,
Staudinger and Roberts 1996):
 
1
Hv;i =R  T1
ki;T2 D ki;T1 e T1 2 ; (13.7)

where ki is the given rate constant and Hv,i the corresponding enthalpy of
volatilization (J mol1 ), and R the gas constant (J mol1 K1 ). We assume that
the key determinant of the liquid-phase rate constant k1 is volatilization from liquid
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 367

to gas phase, while the lipid-phase rate constant k2 is determined by volatilization

of given monoterpene from lipid to liquid phase. Thus, we used the enthalpies of
volatilization for Henry’s law constant (H, gas/liquid phase partition coefficient) for
temperature dependence of k1 and water/octanol phase partition coefficient (Kw/o )
for k2 . Values for ’-pinene (36.9 kJ mol1 for H and 21 kJ mol1 for Kw/o ) were
employed for the sum of monoterpenes emitted (Copolovici and Niinemets 2005).

13.2.2 Scaling Up Models from Leaf to Canopy

in Mediterranean Evergreen Sclerophyll Quercus ilex

13.2.2.1 Canopy Model

The leaf-level emission models were upscaled to an entire stand with the canopy
gas-exchange model GASFLUX (Caldwell et al. 1986; Tenhunen et al. 1994;
Reichstein 2001). In this model, the canopy is divided into homogeneous horizontal
layers, and for each layer light interception and energy balance is modelled, allowing
us to determine leaf temperature and incident photosynthetic photon flux density (Q)
at specific canopy depths from above-canopy meteorological drivers. Each layer
is further split into sunlit and shaded fractions, and leaf gas-exchange rates are
computed according to the biochemical model of foliar photosynthesis of Farquhar
et al. (Farquhar et al. 1980; Harley and Tenhunen 1991). The stomatal conductance
to water vapour (g) is directly bound to net assimilation rate (A) via the Ball-
Berry equation (Ball et al. 1987; Collatz et al. 1991), allowing for simultaneous
estimation of A, g and intercellular CO2 concentration. The computed CO2 , water
vapour and monoterpene exchange is summed over the layers to obtain an integrated
estimate of canopy gas-exchange. The model does not include turbulence of
air, and atmospheric CO2 concentration is taken constant throughout the entire
canopy. The model GASFLUX was parameterized with extensive leaf- and canopy-
level physiological data for Q. coccifera and Q. ilex (Tenhunen et al. 1990;
Reichstein 2001).
All the monoterpene emission models were embedded in the layered canopy
model using first the independent dataset for parameterization of various model
approaches at the leaf scale (Sect. 13.2.2.2) and then embedding these into the
canopy model by various approaches, thereby leading to different model versions
(Sect. 13.2.2.3).

13.2.2.2 Parameterization and Validation Datasets

As an independent parameterization dataset, we used enclosure experiments carried

out on six different Quercus ilex leaves belonging to two different plants grown in
50 L pots (Ciccioli et al. 2001). By measuring the basal monoterpene emissions and
CO2 assimilation rates from leaf development to leaf abscission from May 1997
368 Ü. Niinemets et al.

to September 1998, strong seasonal variations in both parameters were observed

(Ciccioli et al. 2001). From these data, we have determined ES and temperature
and light response curve parameters (Guenther et al. model), ©Tref (ETR model)
and its temperature dependence, rC and its light and temperature dependencies
(C-ratio model, Box 13.1), I and its temperature and light dependence (dynamic
model), and seasonality dependencies for ES (Eq. 13.1), ©Tref and rC . For ES
seasonality, explained variance (r2 ) was 0.81 for increasing and 0.62 for decreasing
part of Eq. 13.1 with the fitted parameters being a D 124, b D 4,045, c D 19.6,
d D 170.5, and f D 75.0.
The validation dataset for 1997 growing season was obtained using trap-
enrichment relaxed eddy accumulation measurements (REA) (Moncrieff et al. 1997)
in a typical Mediterranean Q. ilex dominated forest at Castelporziano, Rome Italy
(41ı450 N, 12ı 220 E, Fig. 13.1 for site details) as described in detail in Valentini et al.
(1997) and Ciccioli et al. (2003).
The exchange rates of water (H2 O) and carbon dioxide (CO2 ) between the
ecosystem and the atmosphere were also measured with the eddy covariance
technique as detailed in Reichstein et al. (2002b, Fig. 13.1). The eddy flux data were
used to test the validity of the GASFLUX canopy model for calculating CO2 and
H2 O canopy gas-exchange. Overall, the canopy model performance was genuine,
and suggested that the parameterization was good and that the basic assumptions
of the model were fulfilled. The explained variance was 79 % for the CO2 flux and
75 % for the water vapour flux, i.e., at the upper limit of what is possible to achieve
with the upscaling models fitted to eddy covariance data. Even neural network
models do not result in considerably higher r2 -values (Wijk and Bouten 1999; Simon
et al. 2005; Boissard et al. 2008). The water vapour fluxes were described slightly
worse than CO2 fluxes by the model, probably because of additional errors resulting
from missing descriptions of the evaporation of intercepted water and from soil
surface in the model.

13.2.2.3 Tested Leaf-Level Model Versions

Qualitatively different approaches were used to parameterize the four different

models, and thus, in the final analysis, we distinguish eight model type/version
combinations that all resulted in different model predictions:
(1) Original parameterization of Guenther et al. (1993) with fixed shapes for
the temperature and light response curves, i.e., cT1 D 95,000 J mol1 ,
cT2 D 230,000 J mol1 , TM D 314 K for temperature response curve, and
’ D 0.0027 mol mol1 , and cL1 D 1.066 for light response curve (Table 13.1
for symbol definition). This set of values has initially been obtained from
measurements of isoprene emission in Eucalyptus globulus, Liquidambar
styraciflua, Mucuna pruriens, and Populus tremuloides, but has also been
demonstrated to provide reasonably good fits to monoterpene emissions
from the leaves of Q. ilex, but with larger deviations at higher temperatures
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 369

150

Precipitation
a

−1
100
50
0
50
b
30

10

−10

−30
c
40

20

0
10
d
−2 −1

0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Fig. 13.1 Meteorological conditions and observed ecosystem monoterpene emissions at the
Castelporziano site in 1997. (a) Daily precipitation above the canopy; (b) daily mean (black
line) and range (grey bars) of air temperatures above the canopy; (c) daily mean (black line) and
range (grey bars) of water vapour pressure deficit above the canopy; (d) total canopy monoterpene
emission rate estimated by trap-enrichment relaxed eddy accumulation (REA). The site (41ı 450 N,
12ı 220 E) is situated 20 km south-west from the centre of Rome, and is an example of climax
forest vegetation of the Mediterranean region with the canopy mainly composed of Quercus ilex,
and associated (<10 %) Q. suber trees. The suppressed understory is dominated by Arbutus unedo
L. and Pistacia lentiscus L. which do not emit monoterpenes. The stand is ca. 10 m tall and has a
leaf area index of around 3.5 m2 m2 . The soil originates from aeolic sands without rocks, and the
roots of canopy trees likely reach ground water (Valentini et al. 1992; Manes et al. 1997a), thereby
escaping the severest summer drought

(T > 35 ı C) (Bertin et al. 1997; Ciccioli et al. 1997) and with positive
or negative deviations for the light response depending on leaf growth
environment, in particular, high vs. low growth irradiance (Staudt et al. 2003).
(2) Fully optimized Guenther et al. (1993) model. In the case of full optimization,
not only ES was parameterized, but also the shapes of the light and temperature
response curves. As data on within-canopy variations in the emission potential
(for within-canopy changes Niinemets et al. 2002a, 2010b) were not available,
370 Ü. Niinemets et al.

models (1) and (2) were initially run with the constant leaf-level parameteri-
zation for all leaves in the canopy. This is different from the C-ratio and ETR
models where within-canopy variation in rC,S and ©Tref results from changes in
assimilation potentials within the canopy. Thus, an inverse modelling approach
was used to find the canopy-level ES,max (Eq. 13.1) value that matched best the
observed canopy monoterpene flux after scaling with the canopy flux model.
A least-squares algorithm was used to perform this model inversion (Visual
Numerics 1993).
(3) Standard parameterization of the ETR model with seasonality (ETR
model C seasonality) where the parameters of T-dependence of © (Table 13.1)
were set to best approximate the relationship between © and T in Niinemets
et al. (2002c) and seasonality in ©Tref was considered as explained above (similar
to Eq. 13.B3). A non-linear fit to estimates of © in Q. coccifera and Q. ilex in
Niinemets et al. (2002c) data gave a Q10 (© at T D Tref C 10 relative to © at Tref )
value of 3.15 (r2 D 0.77, P < 0.001).
(4) In the second model modification (ETR model C fitted temperature depen-
dence), the parameters of the temperature dependence of © (Table 13.1) were
optimized by a least squares algorithm to match the relaxed eddy accumulation
fluxes measured here. Thus, in this model version, temperature dependence
includes both instantaneous and seasonal temperature effects on © as is often
used in modelling VOC emissions (Niinemets et al. 2010c for a review and
critique).
(5) The C-ratio model was applied as explained above (Table 13.1, Box 13.1). The
contributions for current-year leaves and 1-year-old leaves (Fig. 13.B2) were
averaged with equal weights throughout the year and for all canopy layers. The
linkage of monoterpene emissions to foliage photosynthetic characteristics in
models 3–5 might be considered inconsistent, given that relatively weak corre-
lations may occasionally be observed between CO2 exchange and monoterpene
emissions, especially when the measurements in stressed and non-stressed
conditions are pooled (Loreto et al. 1996; Niinemets et al. 2002a). Nevertheless,
it is important to recognize here that the way this is done in these models is
already considering modifications in the fraction of electrons and carbon going
in monoterpene synthesis as affected by temperature and seasonality. Appli-
cation of the ETR- and C-ratio models requires parameterization of the entire
biochemical photosynthesis model of Farquhar et al. (1980) for calculation of
the photosynthetic electron transport rate and gross assimilation rate from the
flux measurements or predictions of net assimilation. This parameterization is
available for the Q. ilex forest in Castelporziano (Reichstein 2001).
(6) Optimized dynamic model, where the standard temperature and light dependen-
cies of Guenther et al. model (model 1) were used for monoterpene synthesis
and the capacity for monoterpene synthesis was fitted by minimizing the sum
of the squares between REA fluxes and model predictions.
(7) Dynamic model running with the rate of synthesis set equal to the input from
the optimized Guenther et al. model (model 2).
(8) Dynamic model with the input from the ETR model C seasonality (model 3).
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 371

In models (7) and (8) it is assumed that the rate of monoterpene synthesis can be
approximated by the steady-state modelled rate of emission. Models (6)–(8) were
used only to simulate the diurnal dynamics. The capacity of the dynamic model to
simulate seasonal variations was not analysed.

13.2.3 Modelling Landscape-Level Monoterpene Fluxes

in a Mixed Hemiboreal Forest

Landscape-level monoterpene emission simulations were conducted at the mixed

hemiboreal forest at Järvselja, south-eastern Estonia (58ı 250 N, 27ı 460 E) (for
detailed site description Noe et al. 2011, 2012). The site is a mosaic of different
vegetation types, mainly resulting from differences in soil fertility, but also due to
forest management (Fig. 13.5a) and has different coverage of dominating evergreen
conifer species Scots pine (Pinus sylvestris, Fig. 13.5b) and Norway spruce (Picea
abies, Fig. 13.5c). In the site, turbulent ecosystem gas-exchange by means of eddy
covariance and measurements of ambient concentrations of reactive trace gases such
as ozone and nitrogen oxides (NOx ) are carried out within and above the canopy
(Noe et al. 2011, 2012).
The emission factors for P. sylvestris and P. abies were estimated from branch
enclosure measurements during field campaigns in summer 2008 and 2009, and
standard emission factors ES , their temperature and light dependencies and cor-
responding foliage gas-exchange rates were derived (Noe et al. 2011). We follow
here the approach of Bäck et al. (2005) and Kulmala et al. (2013) and assume
that the monoterpene emissions in these species result from immediate synthesis,
i.e., are both light- and temperature-dependent as in Q. ilex. This is contrary to
several past studies that have assumed that monoterpenes in these species are
emitted only from storage structures and only depend on temperature (e.g., Simpson
et al. 1995). In reality, the emissions in conifers come both from storage tissues
and from immediate synthesis (Shao et al. 2001; Komenda and Koppmann 2002;
Niinemets et al. 2010c) whereas the emissions relying on immediate synthesis
may even dominate the emissions (Niinemets et al. 2010c). Past studies may have
overestimated the contribution of storage emissions due to “rough handling” of
branches during measurements (Niinemets et al. 2011 for a discussion). Use of
the light-sensitive emission algorithm is consistent with the experimental data at
the site demonstrating that light availability throughout the canopy is a main factor
determining daytime monoterpene flux (Noe et al. 2012).
In the case of our measurement setup with large branches, we were unable to
conclusively separate the different emission sources, although the emissions from
darkened branch cuvettes were small (Noe et al. 2011). Thus, we simulated here
the emissions only for the light period using the Guenther et al. (1993) isoprene
algorithm as for Q. ilex using either the fixed response curve shapes (model 1,
Sect. 13.2.2.3) or responses optimized to the data (model 2). The upscaling of
emission fluxes at different pixels (30  30 m) was done with a simple canopy model
372 Ü. Niinemets et al.

as for Q. ilex using the leaf area index maps (Fig. 13.5b, c), the percentage share of
the species in each grid cell, and incident light and temperature measurements.

13.3 Comparison of Different Model Algorithms

13.3.1 Model Validation Statistics

A number of validation statistics has been used to assess the goodness of model
fit. Traditionally, explained variance of the linear regression between measured
and predicted variables (r2 ) is used to assess how well a model explains the data.
However, r2 measures the association (correlation) between modelled and observed
data, and is thus not sensitive to systematic model over- or underestimation.
A number of other model validation statistics has been proposed (Mayer and Butler
1993; Janssen and Heuberger 1995; Willmott and Matsuura 2005; Moriasi et al.
2007). Nash-Sutcliffe modelling efficiency (Nash and Sutcliffe 1970) is one of most
widely used model validation statistics (Mayer and Butler 1993; Krause et al. 2005;
Moriasi et al. 2007):

P
n
.yi  Pi /2
iD1
NE D 1  ; (13.8)
Pn
.yi  y/2
iD1

where yi is the i-th measured, and Pi the corresponding simulated value of the
characteristic, and y is the mean of the measurements. The modelling efficiency is
an estimate of both the correlation and the coincidence of measured and simulated
values, and as such, is sensitive to systematic deviations between modelled and
observed values (Smith et al. 1996). NE varies from 1 (perfect fit to the data) to
negative infinity (no correspondence with the measurements). A value of NE < 0
indicates that the mean of the measurements is a better predictor than the model.
A disadvantage of NE is that the deviations are calculated as squared values, and
therefore, NE is more strongly influenced by larger values of yi and Pi than by
smaller values (Krause et al. 2005).
Additional model evaluation statistics commonly used are the mean absolute
error:

1X
n
A D jyi  Pi j; (13.9)
n iD1

and the root mean squared error:

13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 373

v
u n
u1 X
Dt .yi  Pi /2 : (13.10)
n iD1

Both these statistics evaluate the average deviation of predicted values from
observations. Both ¢ A and ¢ can be normalized by the range of observations (e.g.,
normalized mean absolute error) or by the average of the observations, thereby
allowing one to compare different datasets. The mean absolute error has been
proposed as a better estimate of model performance than the root mean squared
error, because ¢ can be biased more by numerically larger values of yi and Pi
and also by n0.5 (Willmott and Matsuura 2005). This disadvantage can be partly
compensated by normalization by the standard deviation of the sample (Moriasi
et al. 2007):

NSD D s : (13.11)
P
n
1
n .yi  y/ 2
iD1

Although model comparison studies typically report only a single validation statis-
tic, or in exceptional cases, a few, it is important to recognize that different model
statistics provide complementary information about model performance (Krause
et al. 2005; Moriasi et al. 2007). Therefore, we recommend to provide always
several model validation statistics, including r2 , NE , ¢ A , ¢ and perhaps also ¢ NSD
and range-normalized ¢ A and ¢ to enable comparisons of model applications in
different situations.

13.3.2 Performance of Different Leaf-Level Algorithms

in Simulating Canopy Monoterpene Emissions
in Quercus ilex

13.3.2.1 Simulation of the Diurnal Monoterpene Fluxes

All models (Sect. 13.2.2.3) provided realistic description of monoterpene emission

from the Quercus ilex forest at Castelporziano in the standard day with moderate
model-to-model differences (Figs. 13.2 and 13.3), and the model validation statistics
were similar for different models with optimized Guenther et al. (model 2) and
optimized dynamic model (model 6), yielding the greatest values of modelling
efficiency (Eq. 13.8) and lowest estimates of model deviation (Eqs. 13.9, 13.10 and
13.11, Table 13.2).
Differently from these two models, the Guenther et al. model with only modified
emission factor (model 1) strongly overestimated the emissions in the morning and
in the afternoon (Fig. 13.2). If the light and temperature responses were correct,
 374

Table 13.2 Validation statistics of different monoterpene emission models against observed trap-enrichment relaxed eddy accumulation (REA) fluxes during
the standard day (Figs. 13.2 and 13.3) in evergreen sclerophyll Quercus ilex stand at Castelporziano
Algorithma
Model 1 Model 2 Model 3 Model 4 Model 6 Model 7 Model 8
(Guenther (Guenther et al., (ETR, (ETR C fitted Model 5 (Dynamic, (Dynamic with (Dynamic with
Validation statistic et al., standard) optimized) seasonal ©) ©(T)-function) (C-ratio) optimized) Model 2 input) Model 3 input)
Explained variance (r2 ) 0.83 0.89 0.95 0.89 0.85 0.94 0.90 0.91
Modelling efficiency 0.83 0.87 0.79 0.78 0.76 0.89 0.63 0.50
(NE , Eq. 13.8)
Mean absolute error 0.42 0.33 0.50 0.55 0.65 0.26 0.89 1.20
(¢ A , Eq. 13.9)
Root mean square error 0.65 0.61 0.70 0.73 0.77 0.51 0.94 1.10
(¢, Eq. 13.10)
¢ to observed standard 0.42 0.39 0.45 0.47 0.49 0.33 0.61 0.71
deviation ratio
(¢ NSD , Eq. 13.11)
a
Model details are reported in Sect. 13.2.2.3
Ü. Niinemets et al.
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 375

1500 30

−1
28
1000

−2
26
500 Light
24
Temperature

0 22

6
−1
−2

4
Measured

0
7 9 11 13 15 17 19

Fig. 13.2 Diurnal variations in light and temperature and corresponding modelled and observed
diurnal course of monoterpene fluxes from the Quercus ilex stand at Castelporziano (Fig. 13.1
for site details). The potential of various monoterpene emission algorithms to simulate diurnal
variability in E was compared using the emission rates and meteorological conditions for July 31,
1997 that was a representative day for the 1997 growing season. The models tested (Sect. 13.2.2.3)
were the fully-optimized Guenther et al. model (model 2), standard ETR parameterization
(model 3), C-ratio model (model 5) and optimized dynamic model (model 6). Monoterpene
emission flux was measured using a relaxed eddy accumulation method (Sect. 13.2.2.2)

such an overestimation in the morning can reflect delayed diurnal upregulation of

MEP/DOXP pathway activity as has been observed before (Rasulov et al. 2009,
2010). On the other hand, evening overestimation can be related to constrained
foliage physiological activity due to a decrease in mid-day and afternoon stomatal
conductance that characteristically occurs in Mediterranean habitats during summer
drought (Tenhunen et al. 1987; Reichstein et al. 2002a). Such depressions in
monoterpene emissions have been often observed (Bertin et al. 1997; Moncrieff
et al. 1997; Staudt et al. 1997; Ciccioli et al. 1999). With an improved algorithm
that includes effects of intercellular CO2 concentration (Wilkinson et al. 2009;
Monson 2013 in this volume) such effects of drought can be potentially empirically
accounted for.
Alternatively, morning overestimation of emissions can reflect non-specific
storage of monoterpenes (Ciccioli et al. 1997; Niinemets and Reichstein 2002;
Niinemets et al. 2010c). In the morning, the non-specific pools are small, and thus,
the buildup of non-specific storage reduces the emissions. However, non-specific
storage effects cannot explain the afternoon overestimation.
376 Ü. Niinemets et al.

Fig. 13.3 Simulated vs. a

measured monoterpene Guenther
emission rates from the 6
foliage of Q. ilex in
Castelporziano through the
standard day (the same 4
simulation as in Fig. 13.2).
Canopy monoterpene
emission flux by REA 2

−1
measurements was correlated
1:1

−2
with estimates by (a),
Guenther et al. model with 0
only fitted ES (model 1) and
Guenther et al. model with all
parameters optimized (model b
2), by (b) standard ETR 6
model (model 3) and C-ratio
model (model 5), and by (c)
fully optimized dynamic 4
model (model 6) or dynamic
model using either the input
from Guenther et al. 2
optimized model or ETR
model (model 7). The models 1:1
are explained in Table 13.1
0
and in Sect. 13.2.2.3, and the
model validation statistics are
provided in Table 13.2. In all c
panels, 1:1 lines are also 6
shown
Storage
4

1:1
0
0 2 4 6

−2 −1

Although all the highlighted factors can contribute to the discrepancies between
simulated and observed values, the fit to data by the Guenther et al. model could be
vastly improved by optimizing the light- and temperature responses of monoterpene
emission (Table 13.2, Figs. 13.2 and 13.3). In fact, variations in the shape of
temperature and light response curves for monoterpene emission occur (Staudt et al.
2003; Niinemets et al. 2010a, c), and simultaneously modifying ES and the response
curves is a valid approach. Analogous to the results of these simulations, Keenan and
Niinemets (2012) were able to significantly improve the performance of Guenther
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 377

et al. model in tropical forests. Nevertheless, improving model performance by

simultaneous fit of multiple parameters does not rule out the involvement of other
missing factors. Thus, fitting all parameters simultaneously could potentially lead to
model overparameterization, i.e., in inferior model performance when extrapolated
beyond the measurements using other combinations of environmental drivers that
were not available for model parameterization.
Both the ETR model versions (models 3 and 4) and the C-ratio model (model 5)
performed similarly. In particular, all these models underestimated the emissions
in the morning and in the evening, although the explained variance was high,
especially for the ETR model with seasonal © (model 3). However, all these
models underestimated the emission rate in the morning and in the evening,
reflecting greater light-responsiveness of photosynthesis and, stronger reduction in
photosynthetic activity due to limited stomatal conductance in the evening. As with
the standard Guenther et al. model, rapid increases in the emission rate in response
to increasing light in the morning and rapid reduction in response to decreasing
light and reduced stomatal conductance can lead to overestimates of E and reflect
non-specific storage effects.
Despite differences in parameterization (ETR models 3 and 4) and in the
algorithm complexity (ETR vs. C-ratio model), the similarity in performance
of all “physiological” models is striking, and the results provide encouraging
evidence that with informed parameterization, canopy monoterpene emissions can
be potentially coupled to foliage photosynthetic characteristics, even when using the
simplest algorithms such as the C-ratio model (Box 13.1).
Overall, the best model performance in terms of modelling efficiency and model
deviation was obtained with the dynamic model (model 6) that included non-
specific storage (Table 13.2, Fig. 13.2). This high correspondence between measured
and simulated values was only obtained when the maximum rate of synthesis
(I, Eqs. 13.3, 13.4, 13.5 and 13.6) was separately fitted. In contrast, the worst
correspondence between the predicted and observed values was obtained for the
dynamic model that used emissions from either the optimized Guenther et al.
model (model 7) or ETR model (model 8) as substitutes for the synthesis rate.
In the dynamic model this was improved in the morning and in the evening, i.e.,
reducing the overestimation for Guenther model and reducing the underestimation
for ETR. However, it underestimated maximum fluxes at mid-day, thereby reducing
overall model performance. This comparison clearly suggests that the application of
dynamic emission models requires a separate parameterization of the monoterpene
synthesis component of the model.

13.3.2.2 Seasonal Dynamics of Monoterpene Emission

The seasonal variability in isoprene and monoterpene emission rates (Monson et al.
1994; Bertin et al. 1997, Figs. 13.1d and 13.B1; Staudt et al. 1997, 2000; Guenther
et al. 2000) can result from seasonal changes in the activity of enzymes like isoprene
378 Ü. Niinemets et al.

Table 13.3 Validation statistics of different monoterpene emission models against observed
REA fluxes in evergreen sclerophyll Quercus ilex stand at Castelporziano (Fig. 13.4)
Algorithm
Model 1 Model 3 Model 4
(Guenther, (ETR, (ETR C fitted Model 5
Validation statistic standard) seasonal ©) ©(T)-function) (C-ratio)
Explained variance (r2 ) 0.69 0.74 0.72 0.70
Modelling efficiency 0.67 0.69 0.68 0.69
(NE , Eq. 13.8)
Mean absolute error 0.88 0.95 0.91 0.99
(¢ A , Eq. 13.9)
Root mean square error 1.22 1.22 1.26 1.21
(¢, Eq. 13.10)
¢ to observed standard 0.56 0.47 0.47 0.55
deviation ratio
(¢ NSD , Eq. 13.11)
Model validation statistics and tested models as in Table 13.2. In the case of Guenther et al.
model, the model version with ES,max (Eq. 13.1) derived from the REA flux measurements
by inverse modelling was used (Fig. 13.4a). The performance of dynamic models (models
6–8, Sect. 13.2.2.3) was not analysed

and monoterpene synthases that are responsible for pathway flux (Schnitzler et al.
1997; Lehning et al. 2001). However, temporal changes in enzyme activities may
also be confounded by stress-related decreases in the emission rate (Sharkey and
Loreto 1993; Bertin and Staudt 1996; Staudt and Bertin 1998), e.g., via changes
in the reduced carbon input and limitations due to electron transport or nitrogen
availability. Moreover, changes in the environmental conditions may directly trigger
alterations in enzyme activities (Sharkey et al. 1999; Geron et al. 2000). In the
current study, this complex array of responses was modelled by empirical functions
(Eqs. 13.1 and 13.B3). All models reproduced the relative dynamics well and similar
to the diurnal variability, the models realistically (r2 > 0.69, NE > 0.69) described
the seasonal dynamics of monoterpene emission (Figs. 13.1d and 13.4, Table 13.3).
However, in the case of Guenther et al. model, this high model efficiency was
only achieved when the maximum emission factor (ES,max , Eq. 13.1) was derived
from the REA fluxes by inverse modelling. This may reflect differences in param-
eterization of Guenther et al. and “physiological” models. For the “physiological
models”, the seasonal maximum monoterpene emission rate is determined both
by the photosynthetic activity and either by the fraction of electrons going to
monoterpene emission (©, ETR model) or by the ratio of monoterpene emission
to photosynthesis (rC , C-ratio model). As the photosynthetic activity is described
by a separate model, predicted monoterpene emissions are less sensitive to © or
rC parameterization than is the Guenther et al. model to accurate parameteriza-
tion of ES,max (Eq. 13.1). In fact, when an independent estimate of ES,max from
leaf-level measurements in different plants was used, the Guenther et al. model
significantly overestimated the emissions (Fig. 13.4a, Table 13.3), resulting in low
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 379

10
original ES fitted ES
−1
−2

0
0 5 10 0 5 10 0 5 10

−2 −1 −2 −1 −2 −1

Fig. 13.4 Correlations between the observed (relaxed eddy accumulation, REA, Fig. 13.1d) and
modelled monoterpene emission fluxes from Q. ilex forest at Castelporziano. The simulations in
(a) were conducted with the Guenther et al. model (model 1, Sect. 13.2.2.3) either using an ES,max
value (Eq. 13.1) estimated from the independent parameterization dataset or deriving an ES,max
estimate by inverse modelling using the REA flux data. In (b) the emission flux was simulated by
the standard ETR model with seasonality (model 3) and in (c) by the C-ratio model (model 5). The
1:1 lines are also provided

modelling efficiency (Eq. 13.8) of 0.29 and large mean absolute error (Eq. 13.9) of
1.5 nmol m2 s1 , despite that the explained variance was similarly high (r2 D 0.70)
as for the other models.
The mean absolute difference between the modelled and observed fluxes is
still about one third of the average flux, suggesting that further improvements
of the models, e.g., via more advanced description of underlying physiological
mechanisms, might be necessary. With the current modelling schemes, some
improvement of model predictions may possibly also be achieved by more detailed
parameterization of the vertical variation in foliage physiological characteristics in
the layered canopy model (e.g., Lenz et al. 1997; Niinemets et al. 2010b).
Only the ETR model with seasonality (model 3) and C-ratio model (model 5)
were calibrated independently of the observations. In the other version of the ETR
model (model 4), no seasonality was included, but the temperature dependency
of © was optimized with respect to the observed data, resulting in a very steep
exponential dependency of © on T (Q10 D 7). Given that experimental values of Q10
are below 4 (Niinemets et al. 2002c), the Q10 -value determined from a single fit to
all data is unrealistically high. Because of higher basal emission rates in summer
relative to winter and spring, very high Q10 values are possibly attributable to
confounding effects of temperature and seasonality on ©. Thus, if we had included
the seasonality function as for the other model version, the same Q10 -value of
3.15 derived from the data of Niinemets et al. (2002c) could successfully be
used for the ©(T) function. This underscores the importance of clearly separating
processes that are due to acclimation and lead to changes in the basal emission rate
and instantaneous temperature responses (Niinemets et al. 2010a for an extended
discussion).
380 Ü. Niinemets et al.

13.3.3 Implications of Model Parameterization for Estimating

Landscape-Level Fluxes from a Hemiboreal Forest

Both model approaches, standard Guenther et al. (model 1) and optimized Guenther
et al. (model 2) were applied to estimate the monthly average maximum (between
10 and 14 h) monoterpene emission rate from conifer Pinus sylvestris and Picea
abies dominated ecosystems for July 2010 (Fig 13.5d, e). Picea abies is a more
shade-tolerant species and P. abies dominated pixels supported a greater leaf area
index (LAI) than the pixels dominated by less shade-tolerant P. sylvestris (Fig 13.5d,
e). Guenther et al. model with standard parameters for the light and temperature
dependencies (model 1) led to higher average daytime emissions than the optimized
Guenther et al. model (model 2) (Fig 13.5d, e). The mean normalized deviation
between both parameterizations was about 10 % (Fig. 13.5f), but the spatial
variation in model deviations was large with the greatest deviations found at pixels
with higher LAI. As during the summer months, the daytime temperature remained
most of the time between 22 and 27 ı C, the difference between the models mainly
reflects differences in light parameterization among the two approaches. Especially
at places with a high LAI, the change in light will be more prominent than the
change in temperature. Strong dependence on LAI also implies that in more shade-
tolerant P. abies dominated areas with greater LAI, the deviation among the two
model approaches was greater than in less shade-tolerant P. sylvestris dominated
areas with lower LAI.
Overall, this simulation further emphasizes the importance of accurate parame-
terization of emission models. Differences in model parameterization not only result
in biased site average estimates, but also can importantly alter the spatial distribution
of emissions. While in this simulation, the bias was of the same direction (sign)
across the landscape, both negative and positive deviations can potentially occur for
different parameterizations and for more extended temperature range. This would
lead to false impression of accurate model prediction when testing against integrated
values such as ecosystem-level emission flux measurements by eddy covariance or
REA flux measurements. Thus, comparisons of models at different spatial resolution
can provide important insight into the performance of emission algorithms (e.g.,
Ashworth et al. 2010).

13.4 What Model to Choose: Outlook

13.4.1 What Model Performs the Best?

The model intercomparisons presented here demonstrate that models with widely
differing structure and mechanisms can be successfully parameterized to effectively
predict canopy monoterpene emissions. Once the normalized emission, ES , was
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 381

5 km 10 km 5 km 10 km 5 km 10 km
a b c

0 1 2 3 4 5 6 7 0 1 2 3 4 0 1 2 3 4 5 6 7
Land use codes Pine leaf area index Spruce leaf area index
(m2 m−2) (m2 m−2)

5 km 10 km 5 km 10 km 5 km 10 km

d e f

0 5 10 15 20 0 5 10 15 20 0.0 0.1 0.2 0.3 0.4

Monoterpene emission Monoterpene emission Relative difference
(nmol m−2 s−1) (nmol m−2 s−1)

Fig. 13.5 Comparison of monoterpene emissions at landscape scale. The emissions were pre-
dicted by Guenther et al. model with original static parameterization (model 1, Sect. 13.2.2.3) and
optimized parameterization (model 2) applied to the mixed hemiboreal mixed forest at Järvselja,
south-eastern Estonia (58ı 250 N, 27ı 460 E) (for detailed site description Noe et al. 2011, 2012).
The landcover (a) is defined as: 0 D no data, 1 D productive forest, 2 D low productive forest,
3 D swamps and bogs, 4 D shrublands, 5 D grasslands, 6 D waterbodies, 7 D other (croplands
and suburban habitats). Panel (b) demonstrates the leaf area distribution in Scots pine (Pinus
sylvestris) and panel (c) that for Norway spruce (Picea abies). Leaf area index together with
species-specific emission factors and incident light and temperature were used to simulate average
monthly maximum (10–14 h) monoterpene emission rate in July 2010 according to the original (d,
model 1) and optimized (e, model 2) parameterizations. The difference between the monoterpene
emissions estimates by two different models was scaled to the maximum deviation between both
models and is demonstrated in (f)
382 Ü. Niinemets et al.

correctly described, all models reproduced the seasonal and diurnal dynamics of
emission rate with minor differences among model predictions. The circumstance
that different models have not been necessarily re-parameterized or the scale of
models has not appropriately considered, has been a flaw in many model comparison
exercises. Thus, several past comparisons have not necessarily done justice to some
of the models simply because of inconsistent parameterization (Niinemets et al.
2010c for a discussion). The bottom-line of this intercomparison (Figs. 13.2, 13.3
and 13.4) is that if all models are parameterized in a consistent manner, it becomes
difficult to say which model performs the best.
Overall, the model validation statistics all yielded similar information about the
performance of the model. Nevertheless, r2 was highest for the ETR model that
was significantly biased at lower values of emission rate (Figs. 13.2 and 13.3,
Table 13.2). In contrast, the greatest values of modelling efficiency in the optimized
storage model and optimized Guenther et al. model were associated with the lowest
bias in terms of mean absolute and mean squared error (Figs. 13.2 and 13.3,
Table 13.2). Thus the modelling efficiency together with estimates of model bias
clearly are more informative indicators of model performance than r2 , and they
should be routinely included in BVOC model intercomparison exercises.
Another principal difficulty with testing model algorithms against fluxes inte-
grated or averaged over large spatial areas such as REA fluxes is that such tests are
only valid if vegetation is homogeneous. In the case of non-homogeneous vegetation
(Fig. 13.5) similar fluxes can be predicted with different models, even when the
fluxes are differently distributed across the landscape. However, non-homogeneity
can also amplify the overall deviation if the bias is in the same direction across the
landscape (Fig. 13.5). Thus, analysis of the spatial distribution of deviations among
the models can provide important additional insight into model performance.

13.4.2 What Model to Prefer?

Given the similar performance of different models, we suggest that the preference
of any one particular model over others depends on the availability of data for
model parameterization. The Guenther et al. model is well-established, and its
parameterization requires only monoterpene emission measurements. Moreover,
as implemented in previous studies, many of the required parameters could be
considered as constant for all plants, albeit, as our analysis demonstrates, at the
expense of model predictability (Table 13.2). On the other hand, the model results,
especially for seasonal predictions, are very sensitive to accurate estimation of the
maximum emission factor. Despite this limitation, the Guenther et al. model will be
a preferred model if only emissions need to be calculated and when no information
of foliage physiological activity is available.
From a different perspective, if the monoterpene emission routine needs to be
implemented in a model already predicting stand carbon and water fluxes, linking
13 Scaling BVOC Emissions from Leaf to Canopy and Landscape. . . 383

the emissions to foliage photosynthetic characteristics is recommended. Especially,

because the emission predictions in such models are less sensitive to an independent
estimate of emission capacity, and also may more efficiently capture the effects of
physiological processes such as stomatal closure on the emission rates. The primary
limitation of “physiological” models is that the exact physiological mechanisms
of the regulation of the emission rate are currently still not entirely understood.
Nevertheless, even very simple models such as the C-ratio model (Box 13.1)
performed remarkably well.
The dynamic model that considers non-specific storage of monoterpenes was
one of the best models. The dynamic models qualitatively differ from the other
models by predicting significant night fluxes of emissions and by moderating the
effects of rapid light and temperature fluctuations (Niinemets and Reichstein 2002;
Noe et al. 2006, 2010). The model has been successfully validated at the leaf scale
(Niinemets and Reichstein 2002; Noe et al. 2006, 2010), and although the validation
statistics confirm superior performance of this model at the canopy scale, stand-level
monoterpene fluxes cannot be effectively measured by eddy covariance technique
at night (e.g., Fisher et al. 2007 for a discussion of eddy flux methodology). Also,
the time-resolution of eddy-flux measurements, typically averaged for 30 min. to
reduce fluctuations inherent to eddy technology (e.g., Aubinet et al. 2000) is too
crude to effectively compare the two best models, optimized Guenther (model 2)
and optimized dynamic (model 6). Thus, there clearly are experimental limits for
statistical validation of different models (see also the chapter of Guenther 2013
for further discussion on model comparison). Nevertheless, combining canopy
models to air chemistry models and air reactivity, O3 and NO3 . and OH. radical
measurements may provide indirect ways to validate night emission fluxes (Di Carlo
et al. 2004; Ortega et al. 2007; Sinha et al. 2010).
Given that non-specific storage may be relatively easily implemented in any
emission model, and that it may have a potentially large impact on air chemistry,
we suggest that future canopy-level emission models should consider non-specific
storage. Overall, the existing BVOC emission algorithms are plastic enough to be
parameterized to predict emissions with a high degree of accuracy, and the choice
between different models is not necessarily dictated by inherent differences in
model performance, but rather by practical decisions as driven by the modelling
application. This does not mean that wholly mechanistic models should not be
used when the emission mechanisms become fully elucidated, but simply indicates
the state-of-the-art of BVOC modelling that has reached to a certain level of
convergence of different models (Arneth et al. 2008; Ashworth et al. 2013; Guenther
2013 in this volume).

Acknowledgements Authors work on volatile isoprenoid emission is supported by the Estonian

Ministry of Science and Education (institutional grant IUT-8-3), the Estonian Science Foundation
(grants 8110, 9253), the European Commission through European Regional Fund (the Center
of Excellence in Environmental Adaptation) and European Research Council (advanced grant
322603, SIP-VOLC).
384 Ü. Niinemets et al.

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Chapter 14
Upscaling Biogenic Volatile Compound
Emissions from Leaves to Landscapes

Alex Guenther

Abstract The implementation of biogenic emissions in regional air quality and

global climate models requires numerical code and input datasets that are com-
patible with these regulatory and scientific tools. Canopy- and landscape-level
emission models can be developed using a scaling up approach where emissions
are first calculated on a leaf scale and then scaled up to higher scale using a canopy
model that describes the environmental conditions in different canopy locations.
Alternatively, big-leaf models can be used that simulate canopy emissions based
on the physiological potentials of uppermost leaves in the canopy. Finally, with
development of flux technology for measurement of whole canopy emission fluxes,
canopy-level emission models have been derived that simulate the emissions on the
basis of whole canopy environmental responses. Here the potentials and limitations
of different model frameworks are compared and perspectives for future model
developments are offered.

14.1 Introduction

After several decades of ozone pollution control strategies met with little success,
the US air quality community began to rethink the ozone problem in the late 1980s
(NRC 1991). An outcome of this was a heightened appreciation of the role of
biogenic volatile organic compounds (BVOC) in ozone production. It eventually
became clear that accurate, time varying, gridded BVOC emission estimates were
required for air quality models (Pierce et al. 1998). The demand for quantitative
BVOC emission estimates for global Earth system models has recently been

A. Guenther ()
Biosphere-Atmosphere Interactions Group, Atmospheric Chemistry Division,
Earth System Laboratory, National Center for Atmospheric Research,
3090 Center Green Drive, Boulder, CO 80301, USA
e-mail: [email protected]

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 391
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 14,
© Springer ScienceCBusiness Media Dordrecht 2013
392 A. Guenther

stimulated by studies suggesting a major role of BVOC emissions in controlling

aerosol production (Spracklen et al. 2011; Kulmala et al. 2013 in this volume).
While BVOC emission modelling approaches have improved in the past decades, it
is clear that uncertainties associated with these estimates are still considerable and
may be limiting the development of effective air quality and climate management
strategies.
At the heart of any BVOC emission model are the leaf-level algorithms and
parameters that simulate BVOC emissions from an individual leaf over a wide
range of conditions. As described in earlier chapters (Grote et al. 2013; Li and
Sharkey 2013; Monson 2013), progress in our ability to describe the processes
controlling leaf-level emissions have led to the development of more robust leaf-
level algorithms that can account for a substantial part of the BVOC emission
variations in response to temperature, solar radiation, soil moisture, and ambient
CO2 concentration. These previous chapters also demonstrate that these algorithms
are still not perfect and more work is needed to fully account for the observed
variations in BVOC emissions. In addition, the lack of quantitative algorithms
to account for stress and other factors that are not included in current models
(Niinemets 2010) limits regional model capabilities for quantitatively simulating
BVOC emissions.
Accurate leaf-level BVOC emission algorithms are necessary, but not sufficient,
for estimating the emission inputs needed for regional to global models. The
upscaling from an individual leaf to a whole canopy and then on to an entire
landscape is a challenging task that likely dominates the overall uncertainty in
regional to global emission estimates. Section 14.2 describes the first step in this
process: model approaches for estimating canopy-scale emissions. The second step
discussed in Sect. 14.3 is the characterization of the above-canopy environment.
The final step described in Sect. 14.4, landcover characterization, completes the
task of providing inputs for regional air quality and global Earth system models.
Section 14.5 considers the accuracy of BVOC emission models while Sect. 14.6
address questions associated with similarities and dissimilarities in reported BVOC
emission model estimates.

14.2 Canopy Environment

Most BVOC emissions are sensitive to changes in leaf temperature and some
are also controlled by visible light. Since different leaves within a canopy are
exposed to varying light and temperature conditions, the microclimate of the
canopy environment must be considered for models of canopy-scale emissions. An
obvious implication of the within-canopy variability is that there are much lower
light levels on shaded leaves. This is illustrated in Fig. 14.1 along with another
important consequence which is the higher leaf temperatures on sunlit leaves and the
cooler temperatures on shaded leaves. Given the non-linear light and temperature
responsiveness of most BVOC emissions (Grote et al. 2013; Monson 2013), the
14 Upscaling Biogenic Volatile Compound Emissions from Leaves to Landscapes 393

5 5
4 4

LAI (m2 m−2)

LAI (m2 m−2)

3 3
2 2
1 1
0 0
10 100 1000 294 296 298 300
Leaf PPFD (μmol m−2s−1) Leaf Temperature (K)

Fig. 14.1 Within-canopy variations in incident leaf quantum flux density (PPFD) and temperature
for sunlit leaves (solid line) and shaded leaves (dashed line) on cloudless (thin red lines) and cloudy
(thick black lines) days

overall impact can be complex and varied for different conditions and canopy types.
Of particular importance is that the response of isoprene and other light-dependent
BVOC emissions saturates at high light levels. This means not only that assuming
above-canopy light levels for the whole canopy would overestimate emissions, by
not accounting for the reduced emissions by shaded leaves, but also that the use of
a canopy average light level for the whole canopy would overestimate emissions
by failing to account for the saturated conditions on sunlit leaves. The two basic
approaches used to account for canopy microclimate, bulk (Sect. 14.2.1) and explicit
(Sect. 14.2.2) environment approaches, are described below.

14.2.1 Bulk Canopy Environment Approach

The first BVOC emission models scaled to whole vegetation, including Zimmerman
(1979) and Lamb et al. (1987), used a bulk canopy approach. This entailed using
emission factors and emission response algorithms that were representative of the
average of a range of sun and shade leaves. The canopy was treated as a single
entity with a canopy-scale emission factor and a canopy-scale emission response.
These canopy-scale values were conveniently provided by the branch enclosure
measurement techniques that were commonly used at that time (Zimmerman 1979).
A branch typically includes a range of sun and shade leaves and so represents a
microcosm of the whole canopy. The first temperature and light algorithms were
based on whole-plant measurements made in a gas-exchange chamber (Tingey et al.
1981). These algorithms were therefore representative of BVOC emission response
of a whole canopy, rather than individual leaves, and so were appropriate to apply
using a bulk canopy environment approach, albeit scaling from small plants and
single branches to extensive tall canopies inherently necessitates the use of certain
correction factors to account for greater environmental gradients and within-canopy
variations in emission potentials in mature canopies.
394 A. Guenther

Guenther et al. (2006) used an explicit canopy environment model, with a

leaf energy balance model, to simulate the whole canopy emission response to
light and temperature for a wide range of conditions and canopy types. The
results were used to develop a bulk canopy emission algorithm that was called
the Parameterized Canopy Environment Emission Activity (PCEEA) approach.
This was done to provide modelers with an option with a lower computational
expense. The PCEEA bulk canopy temperature algorithm was similar to the leaf-
level temperature algorithm of the explicit canopy model but was slightly less
sensitive to temperature. This was because the PCEEA bulk approach accounted
for the cooling effect of transpiring leaves. The PCEEA light response algorithm
for isoprene was a function of canopy leaf area index (LAI) and photosynthetic
photon flux density (PPFD). Emissions increased nearly linearly up to an LAI
of 2 but became saturated around an LAI of 3. The PCEEA algorithm increased
emissions nearly linearly with PPFD up to full sunlight in contrast to the leaf-level
light response where emissions saturated at about half of full sunlight.
In addition to branch/plant measurements and explicit canopy model simulations,
a bulk canopy model approach can be based on canopy-scale flux measurements.
For example, Schade et al. (1999) used above-canopy flux measurements to develop
a numerical description of monoterpene response to temperature and humidity.
The increasing availability of above-canopy BVOC flux data has primarily been
used to evaluate explicit canopy models, but should also be used as a resource for
parameterizing bulk canopy environment models.
The bulk-canopy BVOC emission approach is analogous to big-leaf models
of plant photosynthesis (Sellers et al. 1992; Amthor 1994; de Pury and Farquhar
1997), and inherently suffers from potential integration errors due to the spectrum
of different light intensities leaves in the canopy receive at any moment of time. This
can be overcome by using a two-big-leaf approach, where part of the canopy foliage
is sunlit and part is shaded, thereby significantly improving integration of the fluxes
(de Pury and Farquhar 1997; Dai et al. 2004).

14.2.2 Explicit Canopy Environment Approach

The recognition that temporal and spatial variations in canopy structure (e.g.,
leaf area index, species, leaf inclination angles, leaf clumping) and physiological
functioning (e.g., maximal stomatal conductance, photosynthetic capacity) control
carbon, water and energy fluxes has led to the development of explicit models
for quantifying canopy distributions of leaf solar irradiance (e.g., Baldocchi et al.
2002). These models simulate the extinction of solar radiation passing through
the canopy using approaches as simple as assuming a logarithmic decrease with
canopy depth to models that account for leaf orientation, clustering, penumbra
and other effects including three dimensional variability. This subject has been
thoroughly reviewed by Cescatti and Niinemets (2004) and readers are referred
to this paper for a detailed description of these methods and how they have been
14 Upscaling Biogenic Volatile Compound Emissions from Leaves to Landscapes 395

applied to simulate canopy photosynthesis and respiration. This section focuses

on the use of explicit canopy environment models to characterize biogenic VOC
emissions.
Lamb et al. (1993) introduced the first explicit canopy environment model for
estimating BVOC emissions. Their approach was based on the model of Gates
and Papian (1971) that was developed to quantify the solar radiation and energy
budgets of plant canopies at multiple layers in order to estimate canopy-scale
photosynthesis and transpiration. This explicit canopy environment model divides
the canopy into multiple vertical layers and estimates leaf-level solar radiation and
temperature at each level. Since this approach assumes constant light levels across a
given layer, Beer’s law can be applied to assume a logarithmic dependence between
transmission of global solar radiation through the canopy and the product of an
extinction coefficient and LAI as

Qd D Q0 e kLAId (14.1)

where Qd is the solar radiation at a given canopy depth, Q0 is the solar radiation
above the canopy, LAId is the LAI above the given canopy depth and k is an
extinction coefficient that depends on wavelength and canopy structure. Extinction
coefficient is greater for visible (photosynthetic, PAR) radiation and smaller for
near-infrared (NIR) radiation that penetrates deeper into the canopy and thus, for
modelling light effects on BVOC emission fluxes, it is important to partition the
solar radiation flux between PAR and NIR components.
In addition to vertical variations in canopy light distribution, there are substantial
differences in the light levels on leaves at the same canopy depth. Even in the
interior of the canopy, sunlit leaves receive full sunlight due to gaps in the canopy,
albeit often for short periods of time (sunflecks). In contrast, shaded leaves do not
receive direct sunlight. Any given leaf can change from being a sunlit leaf to a
shaded leaf, or the other way around, throughout the day depending on the sun angle
and the location of other leaves and cloudiness conditions. Guenther et al. (1995)
introduced the sunlit and shaded leaf approach for BVOC emission modelling using
the Norman (1982) canopy model which used an approach for estimating the sunlit
foliage portion within each canopy layer and calculating the direct and diffuse
components of solar radiation incident to sunlit and shaded leaves.
Lamb et al. (1993) were the first to use a leaf energy balance model, to calculate
the difference between air temperature and leaf temperature, in an explicit canopy
approach for estimating biogenic VOC emissions. A portion of incoming global
solar radiation, Qabs , is absorbed along with incoming longwave radiation, RIR ,in ,
from the surrounding environment. As shown in Eq. 14.2, this energy is balanced by
outgoing energy which includes sensible heat (RS , convective and conductive heat
fluxes), latent heat (Rœ ) from transpiration and outgoing longwave radiation (RIR ,out )
radiated away from the leaf. Fluxes RS , Rœ , RIR ,in depend on leaf temperature, and RS
and Rœ also on boundary layer conductances for conductive, convective and water
vapour exchange, while RIR ,in depends on surface temperature, in particular, on sky
temperature.
396 A. Guenther

Qabs C RIR;in D RIR;out C RS C Rœ (14.2)

An increase in the incoming energy will increase leaf temperature, and thus outgoing
energy flux, until the leaf energy fluxes are in balance. The numerical calculation of
the leaf energy balance is typically accomplished using iterative numerical solutions
which must be efficient or else will result in substantial computational expense.
There are significant uncertainties in approaches for estimating each of the terms
in the leaf energy balance shown in Eq. 14.2. Qabs is calculated as the difference
between the incoming global solar radiation and the global solar radiation that is
reflected back or transmitted (scattered flux). In addition to the uncertainties in
estimating the incoming solar radiation on a leaf, there are plant to plant differences
in leaf reflectance and scattering coefficients (Goudriaan and van Laar 1994). In
addition, accurate estimation of Qabs requires distribution of solar radiation between
PAR and NIR components and this partitioning importantly depends on cloudiness
conditions that affect the PAR/NIR ratio of global solar radiation (Ross and Sulev
2000). The calculation of RIR,in is dependent on determination of the fraction of the
leaf that is exposed to nearby (1) sun leaves, (2) shade leaves, (3) sky and (4) soil
since each of these objects has a different temperature. Estimating the incoming
infrared radiation from the fraction exposed to the sky requires an estimate of
sky temperature, which is typically not readily available. Leaf sensible heat flux
and latent heat flux calculations require heat transfer conductances that depend on
wind conditions and plant structure and physiological status. Estimations of these
characteristics can introduce some uncertainties (Leuning et al. 1995).

14.2.3 Canopy Model Comparisons

Lamb et al. (1993) compared BVOC emissions estimated using an explicit canopy
model with emissions simply calculated as the product of leaf-level emission factor
and canopy leaf area (no canopy model). They found that isoprene emissions, which
are light- and temperature-dependent, were decreased by a third while monoterpene
emissions, which were only temperature-dependent, were decreased by 6 % (Lamb
et al. 1993). However, if an emission factor based on branch-level measurements
were used with a bulk canopy approach instead of a leaf-level emission factor, there
would be little difference in emissions between the two approaches; this is because
branch-level emission factors are about a third less than leaf-level emission factors
(Guenther et al. 1994).
Above-canopy isoprene fluxes were used to evaluate different canopy models
by Lamb et al. (1996) including (1) a bulk canopy approach, (2) a simple explicit
model (Lamb et al. 1993), and (3) a more detailed explicit model (CANOAK,
Baldocchi and Harley 1995). The Lamb et al. (1993) approach decreases light
levels exponentially through the canopy, while CANOAK accounts for the impact
of leaf clumping on radiative transfer and the influence of turbulent diffusion on
14 Upscaling Biogenic Volatile Compound Emissions from Leaves to Landscapes 397

Fig. 14.2 Comparison of 12

eddy covariance observations Predicted
(grey line) and MEGAN Observed
model estimates (red dots) of 10

Isoprene Flux (mg m−2 h−1)

isoprene emissions from an
Amazon tropical forest 8
canopy. Eddy covariance
measurements were
conducted using 6
proton-transfer reaction mass
spectrometry (PTR MS) and
4
the MEGAN model estimates
were simulated using an LAI
of 6, an isoprene emission 2
factor of 5.9 mg m2 h1
(Modified from
0
Karl et al. 2007) 261 263 265 267 269 271
Day of Year

canopy exchange. The results showed that the fluxes estimated with the three
models were consistent to within about 20 %. The models initially overestimated
the mean flux by a factor of two but could be brought into agreement by adjusting
the emission factor and biomass density to values that were within the uncertainty
range for these factors. It is remarkable that the Lamb et al. (1996) study appears
to be the only comparison of different canopy environment models that includes an
evaluation with above-canopy flux data. The above-canopy isoprene flux dataset
used for the Lamb et al. canopy model comparison consisted of only about 80
relaxed eddy accumulation (REA) and gradient flux measurements (Guenther et al.
1996) which is very small compared to the number of measurements available from
more recent eddy covariance studies (e.g., Pressley et al. 2005). Figure 14.2 shows
an evaluation of modelled isoprene and monoterpene emissions from a tropical
forest canopy (Karl et al. 2007). The results demonstrate the agreement between
model estimates and eddy covariance measurements by a proton-transfer reaction
mass spectrometer (PTR MS). Partial correspondence between measurements and
simulations as demonstrated in this study is often observed when site-specific
parameterizations are available (Niinemets et al. 2013). While the model describes
the general observed behavior, there are a number of details that are not captured
by the model. Overall, we note that it is difficult to achieve full correspondence
between the model and measured estimates due to inherent uncertainties in both
model algorithms and parameterization and in BVOC flux measurement techniques
(Niinemets et al. 2013).
Guenther et al. (2006) conducted a sensitivity study with a range of input values
using a simple (BEIS, based on Lamb et al. 1993 and updated by Pierce et al.
1998) and a more detailed (MEGAN, based on Guenther et al. 1995 and updated
by Guenther et al. 1999) explicit canopy environment model. The differences
were typically within 30 % but were greater in some cases. The impact of
398 A. Guenther

different explicit canopy environment models on estimated isoprene emissions was

investigated by Keenan et al. (2011) who found differences exceeding a factor of
two and concluded that the large differences in estimated sunlit and shaded leaf
area fractions were largely responsible for the observed discrepancies. They also
demonstrated that the difference in estimated emissions was highly dependent on the
leaf emission algorithm used in the comparison. On the other hand, the discrepancies
between different emission algorithms can be greatly reduced by appropriate re-
parameterization of given algorithms (Keenan et al. 2009; Niinemets et al. 2013),
an approach that is often avoided by BVOC modelers who prefer to use “default”
parameterizations.

14.3 Above-Canopy Environment

An accurate simulation of BVOC emissions requires an accurate characterization

of the above-canopy environment. This is usually straightforward for canopy-scale
flux estimates at a specific site, since direct measurements are typically available,
but is considerably more challenging for landscape-scale emission modelling,
especially in regions where few observations are available. The above-canopy
environmental variables needed include wind and humidity, which can influence
leaf temperature, but the most important drivers are soil moisture, solar radiation
and temperature. Weather data for driving BVOC emission models are readily
available from many different sources including interpolated observations (http://
www.cru.uea.ac.uk/data, http://www.metoffice.gov.uk/hadobs/), model predictions
(http://www.cesm.ucar.edu, https://esg.llnl.gov:8443/), and model reanalysis that
assimilate observations by nudging the model simulation towards the observed
value (http://www.esrl.noaa.gov/psd/data/gridded/data.ncep.reanalysis.html, http://
www.ecmwf.int/research/era/do/get/index).
Guenther et al. (2006) examined the sensitivity of global annual total isoprene
emissions by driving a global model with five different temperature and solar
radiation databases and found that global isoprene emissions ranged from 14 to
C15 % of the standard case which used the NCEP reanalysis. More importantly,
they reported regional differences in isoprene emissions of up to a factor of 3 when
using different weather driving variables. Three different global BVOC emission
models were compared by Arneth et al. (2011) who also found substantial regional
differences in isoprene emissions associated with different temperature and solar
radiation inputs. They noted that the impact of changing the weather data differed
among the different BVOC emission models. Guenther et al. (2006) and Muller et al.
(2008) used the same algorithm, but different soil moisture data, to estimate the
influence of soil moisture on annual global isoprene emission. Muller et al. (2008)
estimated a 20 % decrease in isoprene due to the soil moisture effect, while Guenther
et al. (2006) calculated only a 7 % decrease. This was primarily due to uncertainties
in predicting soil moisture. However, even if accurate soil moisture estimates are
available, the exact “soil moisture effect” is difficult to simulate because limited
14 Upscaling Biogenic Volatile Compound Emissions from Leaves to Landscapes 399

soil water availability can initially increase emissions, while a severe drought can
greatly reduce the emissions (Calfapietra et al. 2013; Monson 2013).
Wang et al. (2011) investigated the BVOC emission model uncertainties as-
sociated with weather inputs derived from a regional weather model (MM5)
simulation of the Pearl river delta in China. By comparing the MM5 output with
observations, they determined an average overestimation of 2o C for temperature
and 120 W m2 for downward shortwave radiation. These errors are similar to
the error values typically observed in weather model simulations in the eastern US
(Hanna et al. 2005). Wang et al. (2011) attributed the errors to a lack of aerosol
impacts on solar radiation in MM5. These model input errors were associated with
errors in predicted isoprene emission fluxes of 23 % due to temperature bias and
45 % due to solar radiation bias. The impact on monoterpene emissions was 17 %
due to temperature and 19 % due to solar radiation. Even regional models that
account for aerosol impacts on solar radiation, such as WRF (Grell et al. 2005),
have difficulties in accurately predicting downward solar radiation. This is primarily
due to the challenge of accurately predicting cloud cover as well as difficulties in
evaluation of the scattering characteristics of different types of clouds. Accurate
prediction of global solar radiation is a problem even in apparently “clear-sky”
conditions. Guenther et al. (2012) compared North American isoprene emissions
estimated using solar radiation simulated by WRF and solar radiation measured
by satellite. The WRF driven estimates were overestimated by 37 % even for
“clear sky” cases. They concluded that WRF could not resolve the thin high-
level baroclinic shield of cirrostratus or altostratus occurring at 6–9 km above sea
level. The situation is even more complicated under cloudy and partially cloudy
conditions.
Because the BVOC emission response to temperature and solar radiation is
non-linear, BVOC emissions are sensitive to the temporal and spatial resolution
of weather input data. For example, the temperature response of most BVOC
emissions is exponential. The arithmetic average temperature will underestimate
emissions and neglect the high emissions that occur during even a short period of
high temperature (e.g., Niinemets et al. 2011). Ashworth et al. (2010) found that
global annual isoprene is reduced by 3 % when using a daily average temperature,
and 7 % when using a monthly average temperature, instead of using an hourly
average temperature. The impact was much greater on local scales with reductions
of up to 55 % when using monthly rather than hourly data (Ashworth et al. 2010).
Correspondence between spatial and temporal scales is a key issue in modelling
(Jarvis 1995) that still receives less consideration than it deserves.
Coarse spatial resolution can also introduce errors due to arithmetic averaging.
This was tested by running the MEGAN model (Guenther et al. 2012) over a
mountainous domain in eastern Tennessee and western North Carolina in the
US. Figure 14.3 shows that the variations in elevation in this region led to large
temperature differences, and thus, emission activity differences of more than a factor
of four, and yet increasing the spatial resolution from 100 to 1 km2 had a fairly
small impact (4 %) on isoprene emissions when a constant landscape average
emission capacity was assumed. However, Fig. 14.3 shows that the landscape in
400 A. Guenther

Temperature
emission
Tennessee Emission Tennessee
capacity
activity River Valley River Valley
(mg m−2h−1)

Blue Ridge Mtns,

Blue Ridge Mtns,
North Carolina
North Carolina

Fig. 14.3 Temperature- and landcover-driven variations in isoprene emissions in a region of

variable elevation along the Tennessee and North Carolina, US border for July 2000. The emissions
were simulated with MEGAN2.1 (Guenther et al. 2012)

this region is not homogeneous with respect to isoprene emission capacity. The
higher (and cooler) elevations tend to have a much higher fraction of oaks which
are high isoprene emitters. As a result of the negative correlation between low
temperatures with high isoprene emission capacities, there was a 12 % decrease in
isoprene emission when spatial resolution was increased from 100 to 1 km2 . An even
greater decrease (20 %) in total BVOC emissions with increasing spatial resolution
was estimated for central Colorado, USA, where the higher (cooler) elevations are
covered by higher emitting forests and the lower (warmer) elevations support lower
emitting grasslands.
BVOC emission models are typically driven by a downward solar radiation value
from a model or observation. If an explicit canopy model is used, then emission
estimates are sensitive to the decomposition of the above-canopy solar radiation into
direct vs diffuse and PAR vs NIR components. The uncertainties in the values used
to parameterize radiative transfer above and within the canopy make a significant
contribution to the overall uncertainties in BVOC emission estimates (Guenther
et al. 2012). For example, BVOC algorithms typically require solar radiation inputs
in units of mol photons m2 s1 . Since atmospheric values are typically in units of
W m2 , a conversion factor between quantum and energy units is required. Reported
values for different sites and conditions range from less than 4 to greater than
5 mol J1 , and accurate conversion factor cannot be derived without information
of solar radiation spectrum (Ross and Sulev 2000). This uncertainty in the PPFD
conversion factor leads to an uncertainty in isoprene emissions of about ˙13 %.
In addition, the value for diffuse PPFD, especially for clear-sky conditions, is
considerably less than that for direct PPFD. MEGAN2.1 (Guenther et al. 2012)
accounts for this by using different values, 4.6 mol J1 for direct PPFD and
4.3 mol J1 for diffuse PPFD. As shown in Fig. 14.4, the decomposition of PPFD
into direct and diffuse fractions is also difficult. The updated approach of Guenther
et al. (2012) in MEGAN2.1 yields 10–50 % higher estimate of diffuse PPFD than
that of Guenther et al. (2006) under cloudy skies and more than a factor of two
14 Upscaling Biogenic Volatile Compound Emissions from Leaves to Landscapes 401

Fig. 14.4 Comparison of 600

diffuse quantum flux density

PPFD Predicted (μmol m−2 s−1)

(PPFD) observed at Boulder, BEIS3.14
CO, USA on June 28, 2008 500 MEGAN2
and predicted using model MEGAN2.1
approaches of BEIS3.14 400
(Based on Pierce et al. 1998),
MEGAN2 (Guenther et al.
300
2006), and MEGAN2.1
(Guenther et al. 2012). Solid
black line indicates 1:1 200
agreement between the
observed and predicted 100
diffuse quantum flux density

0
0 100 200 300 400 500 600
PPFD Observed (mmol m−2 s−1)

higher estimate under clear-sky conditions. A higher fraction of diffuse light can
increase isoprene emissions by increasing light penetration to shade leaves, as at
any moment of time, diffuse light can penetrate through all gaps in the canopy,
while direct light only though the gaps that are on the solar beam path. However,
since the Guenther et al. (2006) leaf-level algorithms assume that shade-adapted
leaves are not very responsive to increases in PPFD, a 25–50 % increase in diffuse
PPFD results in only 5–10 % increase in isoprene emissions under cloudy skies and
a factor of two increase in diffuse PPFD under clear skies results in only 5 %
increase in emissions. The impact could be greater using other canopy environment
models. For further discussion on the global modelling uncertainties the reader is
referred to the chapter of Asworth et al. (2013) in this volume.

14.4 Landcover

The pioneers of BVOC emission modelling had few options for obtaining landcover
data for estimating regional- to global-scale BVOC emissions. The available scaling
approaches consisted of simply multiplying a branch-level emission to a rough
estimate of the regional or global total biomass (e.g., Rasmussen and Went 1965).
The resulting emission estimates are surprisingly similar to the output of current
models that use detailed emission algorithms, emphasizing the important role of
amount of biomass in determining canopy, region and global estimates of BVOC
emission.
BVOC emission models have assumed that foliage is the dominant BVOC
source and scaled BVOC emissions to an estimate of the amount of foliage, either
LAI or foliage mass per ground area. Foliage mass was used in earlier studies
because it was easier to measure. As a result, most leaf and branch emission
402 A. Guenther

measurement data were normalized to leaf dry weight. Representative data on

standing foliage biomass were available for broad ecosystem types and constant
values were assigned based on literature compilations (e.g., Box 1981). Seasonal
variations in foliage amount could be predicted using empirical algorithms driven
by precipitation and temperature (e.g., Lieth and Box 1977). The availability of
satellite observations of ecosystem greenness in the late 1980s provided a potentially
better alternative for quantifying LAI variations within a given vegetation type. The
initial data based on Advanced Very High Resolution Radiometer (AVHRR, http://
nsidc.org/data/avhrr/), a weather satellite, had relatively large uncertainties and so
they were used to drive monthly variations, but the peak LAI was still a constant
assigned to each ecosystem type (e.g., Guenther et al. 1995).
The deployment of improved satellite landcover sensors and several decades
of refining satellite algorithms provides more confidence in satellite landcover
products although considerable uncertainties remain especially due to saturation
of reflectance-based information at relatively low LAI values and difficulties in
estimating spatial aggregation of foliage from remote sensing products. Current
global LAI products include the NASA MODIS data (http://modis.gsfc.nasa.gov/
data/) and the ESA SPOT/VEGETATION data (http://www.spot-vegetation.com).
Garrigues et al. (2008) compared these two products with ground observations and
found that each product performed better in some ways. SPOT generally agreed
better with observations from lower LAI ecosystems such as shrublands and savan-
nas, but MODIS estimates were superior in high LAI ecosystems such as forests.
MODIS is available globally for 2003 to present while the SPOT LAI is currently
only available from October 2009 to present. A significant advantage of remote
sensing products over other approaches is the potential to characterize LAI changes
associated with disturbances and later regrowth. For example, Fig. 14.5 illustrates
satellite-based observations of the decreased LAI associated with wildfires and bark
beetle outbreaks in Colorado. Satellite derived seasonal variations in LAI should
also be an improvement over empirical algorithms, e.g., in wet tropical regions
where light limitations may be more important. Although initial satellite databases
provided monthly average data, higher-resolution data, e.g., 8-day data, are now
widely available. However, the higher-resolution data availability will depend on
cloudiness and air clearness conditions in given areas, limiting the use of these data
for some areas or for some periods such as periods of rain or vegetation burning.
Monthly and ten day average MODIS LAI data are compared in Fig. 14.6. The 8-
day data often appear to be relatively noisy and so may not present a significant
advantage over monthly data. However, Fig. 14.6 also shows that the 8-day data
capture some features in landscapes dominated by deciduous vegetation, especially
fast growing plants such as some crops.
Regional to global land surface models quantify the variability in landscape
characteristics by using either a landcover approach or a plant functional type
(PFT) approach. The landcover approach categorizes entire landscapes (e.g., mixed
forest, savanna, mixed woods and urban) while the PFT approach categorizes the
individual components that occur within a landscape (e.g., temperate cool conifers,
14 Upscaling Biogenic Volatile Compound Emissions from Leaves to Landscapes 403

Fig. 14.5 Percent change in July 1–8 leaf area index (LAI) of Colorado Rocky Mountain forests
between years 2003–2005 compared to years 2010 and 2011 based on MODIS satellite data
products used as MEGAN2.1 input data (Guenther et al. 2012). Areas of increased LAI may
indicate forest regrowth after wildfires, while areas of reduced LAI may indicate areas impacted
by mountain pine beetle (Dendroctonus ponderosae) or other disturbances. State borders and city
names are shown for reference

C4 grasses). Guenther et al. (1995) assigned isoprene and monoterpene emission

factors to the 72 landcover types in a global database. Most of these landcover
categories did not represent specific isoprene or monoterpene emission types, for
example, temperate deciduous forests do not all have the same isoprene emission
capacity. Other, e.g., coastal mangrove category with limited number of species,
more closely represented specific isoprene or monoterpene emission categories.
Since the measurement data on isoprene and monoterpene emissions were only
available at locations representing half of these landcover types, there was no need
for a more detailed approach. Wang and Shallcross (2000) converted the Guenther
et al. (1995) landcover type emission factor scheme into the PFT approach that
was becoming increasingly common in Earth system models. The availability of
404 A. Guenther

Fig. 14.6 Comparison of a

monthly (dashed thick grey Oak

LAI (m2 m−2)

line) and 8-day (solid thin 4 Woodland
black line) average leaf area (CO)
index (LAI) for three regions 2
of the western US in year
2008. The leaf area index data
were derived from MODIS 0
satellite data products used as b
San Joaquin

LAI (m2 m−2)

MEGAN2.1 input data 2
cropland (CA)
(Guenther et al. 2012)
1

0
c
Okanogan grassland (WA)
LAI (m2 m−2)

0
0 60 120 180 240 300 360
Day of Year

high-resolution satellite-based landcover data and additional BVOC measurements

enabled some advancement for BVOC emission modelling in the following decade.
Guenther et al. (2006) devised a flexible global framework that integrated the PFT
and landcover approaches with a high resolution (1 km2 ) suitable for regional
modelling. Each location was associated to one of several thousand ecosystem
types. In addition, the fraction of each of 6 PFTs were assigned to each location.
For regions where data were available, quantitative tree inventories were combined
with species-specific emission factors. This was implemented in the model through
gridded emission factor maps for individual compounds for each PFT type (e.g., an
emission factor map for isoprene emissions from broadleaf trees). This approach
combined a large number of landcover and emission measurement data which made
it difficult to reference the source of information for the emission factor assigned to
each location. Guenther et al. (2012) extended this approach to 16 PFTs to make it
more compatible with Earth system models. Since the Guenther et al. (2012) model
also has 19 emission categories, over 300 gridded emission factor maps would
be required to specify emission factors for each emission and PFT type. Future
approaches should extend the PFT scheme to a larger, but limited (<50), number
of emission types. This would facilitate efforts to provide more transparency in
describing the basis for each emission factor. It may also be beneficial to link the
parameterizations to dynamic vegetation models. This would make it possible to
predict changes in emission categories through vegetation succession, e.g., from
greater isoprene emissions in early successional forests towards greater prevalence
of monoterpene emissions in late-successional forests (Harrison et al. 2013).
14 Upscaling Biogenic Volatile Compound Emissions from Leaves to Landscapes 405

14.5 Assessing the Accuracy of BVOC Emission Models

BVOC emission modelling began more than 50 years ago with a simple calculation
that Went (1960) outlined to characterize the potential contribution of BVOC
emissions to petroleum formation. A decade later, Rasmussen (1972) asked the
question “what do the hydrocarbons from trees contribute to air pollution?” and
addressed it by integrating a forest inventory with species-specific emission factors
for isoprene and ’-pinene. The result suggested that biogenic VOC emissions from
just forests were about six times greater than anthropogenic sources. Zimmerman
(1979) and Winer et al. (1982) used branch enclosure emission measurements to
characterize emissions from important North American species and integrated these
into regional BVOC emission estimates. Gridded landcover data were generated
for specific locales (e.g., San Francisco Bay area, southwestern Virginia) and
emission inputs were calculated for ozone model simulations (Salop et al. 1983).
The US EPA began using time varying, gridded BVOC emissions for regional
air quality modelling in 1986 (Pierce and Waldruff 1991). The procedures were
adapted from Lamb et al. (1987) which had a domain limited to the continental
US and included three emission categories: isoprene, ’-pinene and other non-
methane hydrocarbons. Five wildland landcover types were used including oak
forest, other deciduous forest, coniferous forest, scrubland, and grassland. Many
different crop types were included in the model, although they only accounted for
3 % of the estimated emissions. An uncertainty of 210 % (about a factor of three)
was associated with these BVOC emission estimates based on the propagation of
uncertainties in emission factors, emission algorithms, amount of biomass, and
land use distributions. However, this uncertainty estimate neglected many of the
components that are now routinely found in BVOC emission models. For example,
the Lamb et al. (1987) isoprene emission variations due to changes in solar radiation
were simulated by assigning zero emissions at night and assuming constant solar
radiation during the day.
The first US EPA biogenic emission model, called BEIS (Pierce and Waldruff
1991), was released in 1988. The second version of the model (BEIS2), released
in the mid 1990s, predicted dramatically different estimates of isoprene emission
rate, by about a factor of five higher than BEIS (Pierce et al. 1998). BEIS and
BEIS2 differed in many aspects including leaf-level emission algorithms, biomass
densities, and landcover distributions. However, the major driver of the difference
was the emission factors which were based on Guenther et al. (1994) rather than
Zimmerman (1979). Guenther et al. (1994) concluded that the Zimmerman (1979)
measurements underestimated isoprene emission factors through the use of shaded
branches and overestimated monoterpene emission factors due to disturbances
associated with the measurement technique. As discussed in Sects. 14.2, 14.3 and
14.4, there are a number of individual BVOC emission model components that each
contribute uncertainties of 10–30 %. These uncertainties, if they are all in the same
direction, can add up to a factor of two or more. And yet, the emission factor remains
406 A. Guenther

the dominant contributor and can result in uncertainties of a factor of five or greater
in regions were emissions from the dominant vegetation are not well characterized.
Hanna et al. (2005) used a Monte Carlo probabilistic approach to estimate
uncertainties associated with BEIS3 BVOC emission model outputs and their
impact on regional ozone concentrations. The assessment considered the area-
averaged emission factor which integrates both plant species specific emission
factors and plant species composition, nine emission algorithm parameters, and
three model inputs (LAI, temperature and solar radiation). The 95 % confidence
range on the calculated uncertainty in isoprene emission was about one order of
magnitude, while the calculated uncertainty for monoterpenes and other BVOC
was only ˙20 %. This is contrary to what is expected since our understanding
of isoprene emission is greater than that of other BVOC. The reason for their
assignment of higher uncertainty to isoprene seems to be that there were more
parameters associated with the isoprene emission algorithm. This emphasizes the
need to consider not only the uncertainties due to the factors that are considered in
BVOC emission models, but also the potentially larger uncertainties associated with
processes that are not considered in BVOC emission models.
While comparisons of BVOC emission models provide little information about
the accuracy of these models (see also Niinemets et al. 2013), the availability of
independent observations can inform us. Comparisons of canopy-scale fluxes and
emission models tend to agree within 30 % when site-specific parameters are used
(Lamb et al. 1996), while comparisons of canopy-scale flux measurements when
scaled to regions or globe and compared with regional and global model output
often differ by a factor of two or more (Muller et al. 2008; Arneth et al. 2011). It
should be noted that these are not direct comparisons due to the difference in scale
between a canopy flux measurement and the resolution of a global model.
Aircraft measurements provide the means to directly evaluate BVOC emissions
models and have the potential to dramatically improve assessments of the accuracy
of landscape average emissions. One approach is to use ambient concentration
measurements and infer the fluxes required to maintain the observed concentration
distributions. The limitation of this approach is the requirement for accurately
describing chemical losses and dispersion. Warneke et al. (2010) used an extensive
aircraft database to examine the performance of two BVOC emission models, BEIS3
and MEGAN2. They concluded that MEGAN2 isoprene emissions tended to be
higher than the observations and BEIS3 isoprene emissions tended to be lower, but
both models were within the factor of two uncertainty of the measurement approach.
In addressing the question of whether this should be considered a “good” agreement,
Warneke et al. (2010) point out that anthropogenic emission estimates are often off
by more than a factor of two. Karl et al. (2009) have successfully demonstrated
a PTR MS eddy covariance flux measurement approach that can provide high-
resolution BVOC flux measurements. Aircraft flux measurements systems can be
used to accurately quantify BVOC emission fluxes at the scales required to evaluate
regional and global models.
Satellite-based estimates of formaldehyde distributions over specific regions
have been used to evaluate BVOC emission models (Barkley et al. 2009;
14 Upscaling Biogenic Volatile Compound Emissions from Leaves to Landscapes 407

Stavrakou et al. 2009; Marais et al. 2012). However, the uncertainty associated
with the satellite approach is 40 % for high NOX regions and 40–90 % for
low NOX regions (Marais et al. 2012). In most cases, satellite-based estimates
are within 50 % of BVOC emission models which indicates good agreement,
especially given the uncertainties associated with the two approaches (Stavrakou
et al. 2009; Marais et al. 2012). This also gives us some confidence that regional-
scale isoprene emission estimates are within 50 % of the “true” value, although
there are exceptions (e.g., Barkley et al. 2009).
Our limited ability to quantify the accuracy of BVOC emissions precludes a
detailed quantitative assessment of BVOC emission model uncertainty, but local
canopy flux tower data, regional aircraft concentration distributions, and global
satellite-based emission estimates all suggest that isoprene emission estimates are
usually within a factor of two of the “true” emission flux. Higher uncertainties
are expected for specific locations where the isoprene emission capacities of the
dominant vegetation are unknown and also for regions impacted by stress. The
impact of this uncertainty on the accuracy of ozone simulations is highly dependent
on the chemical regime of a given region. BVOC uncertainties are important in
BVOC-sensitive regions but less important in other areas. The growing recognition
of the role of BVOC in secondary organic aerosol formation will increase the
requirement for more accurate BVOC emission estimates. The best approaches
for accurate assessment of regional BVOC emission rates are based on airborne
direct eddy covariance flux measurements. These measurements have advanced
from relaxed eddy accumulation (Greenberg et al. 1999) and variance (Karl et al.
2004) techniques to direct eddy covariance methods that can be applied at very high
(2 km2 ) resolution (Karl et al. 2009). More widespread application of aircraft eddy
flux techniques, which would benefit from the development of an approach for low
cost light aircraft, could enable verification of BVOC emission models and provide
observations for improving parameterizations.

14.6 Why Are Estimates of Global Isoprene Emissions So

Similar (and Why Is This Not So for Monoterpenes)?

The large difference in BEIS and BEIS2 isoprene emissions, discussed in the
previous section, emphasized the high uncertainty in these model estimates. In
contrast, not all comparisons of BVOC emission models should be expected to
reflect the uncertainty in the emission estimates since the models may be based
on the same or similar parameterizations and approach. Arneth et al. (2008) found
that the annual global isoprene emission estimates reported by 15 studies were
“surprisingly” similar, and yet the monoterpene emission estimates were quite
different, and posed the question “Why are estimates of global terrestrial isoprene
emissions so similar? (and why is this not so for monoterpenes)?”. They noted
that the standard deviation in these isoprene emission estimates was only a little
more than 10 % of the mean value. They recognized that part of the reason was
408 A. Guenther

that all of the models used at least some of the algorithms or emission factors
from Guenther et al. (1995), but they expected larger differences due to the various
driving variables used by the models. Table 1 of Arneth et al. (2008) lists the annual
global emission estimates of the 15 studies and states that the same global value
(503 Tg C year1 ) was reported by both Guenther et al. (1995) and Guenther et al.
(2006). This is incorrect as Guenther et al. (2006) only report a range of values
for different simulations and give an approximate value for their base case which
is 10 % higher than the Guenther et al. (1995) estimate. While not exactly the
same estimate, this is still a small difference and does not significantly change the
standard deviation of the results from the 15 studies.
Arneth et al. (2008) note that the range in the annual global isoprene emission
estimated by the 15 studies, 189 Tg C year1 , is similar to the range reported
by Guenther et al. (2006) for results using a single model with 24 different sets
of driving variables. The variability in the model simulations listed in Table 4 of
Guenther et al. (2006) is 13 % which is similar to that reported for the 15 studies by
Arneth et al. (2008). The answer to the first part of the question seems to be that all
15 studies used the same general approach with similar emission activity algorithms
and emission factors. While driving variables can result in large differences for a
specific location and time, they tend to result in differences of 15 % on the annual
global scale.
In addressing the second part of their question, Arneth et al. (2008) conclude
that “There is no apparent reason that the spread in monoterpene emission rates
should be so much larger compared to isoprene emission rates.” They go on to argue
that BVOC emission modelling is in the “illusion phase: a lack of observations
prevent independent model evaluation and the models have the propensity to not
depart greatly from previously published estimates”, but that the divergence of the
monoterpene emission estimates indicates that monoterpene emission modelling
has advanced to the “chaos phase where model results diverge freely, reflecting
more candidly the lack of observational constraints and of true process modelling”.
Another explanation is simply that, as shown in Fig. 14.7, light-independent
monoterpene emission estimates scale almost linearly with foliar biomass, while
the isoprene emission estimates become saturated at certain LAI due to increasingly
stronger light-limitation on emissions. If we eliminate the three studies reporting
lower monoterpene emission rates (Levis et al. 2003; Naik et al. 2004; Schurgers
et al. 2009) in Table 1 of Arneth et al. (2008) then the variability (9 %) is less
than that for isoprene. The difference between the Levis et al. (2003) and Guenther
et al. (1995) estimates of foliar biomass in tropical forests results in about a factor
of three difference in monoterpene emissions, but a relatively small difference in
isoprene emission.
The difficulty with monoterpene emissions is that there are light-independent
emissions from storage in species such as conifers, and light-dependent emissions
in species lacking storage structures such as many broad-leaved emitting species
(Grote et al. 2013; Niinemets et al. 2013). This is complicated further by the
presence of both emission behaviors in some species (Taipale et al. 2011). Schurgers
et al. (2009) showed that the difference in monoterpene emission algorithm used
14 Upscaling Biogenic Volatile Compound Emissions from Leaves to Landscapes 409

Fig. 14.7 Isoprene and 2

light-independent
monoterpene emission Isoprene
responses to vegetation leaf Monoterpene
area index (LAI) simulated
by MEGAN2. Emission is
normalized by the emission

Relative emission
at a LAI D 5

0
0 2 4 6 8 10
Leaf Area Index (m2 m−2)

resulted in only a 7 % difference in emissions, although clear separation of species

into storage and non-storage monoterpene emitters (Fineschi et al. 2013; Grote et al.
2013; Niinemets et al. 2013) and consistent landcover categories might be difficult.
In addition, differences in emission factors appear to play a small role since Levis
et al. (2003), Schurgers et al. (2009), and Naik et al. (2004) values were also based
on the emission factors of Guenther et al. (1995). There are some apparent errors
in the way in which Naik et al. (2004) and Schurgers et al. (2009) adapted these
emission factors. Guenther et al. (1995) included categories for tropical seasonal
forest and drought deciduous landscapes. The drought deciduous landscape was
intended to represent shrublands but Naik et al. (2004) used this value for tropical
broadleaf drought-deciduous trees and Schurgers et al. (2009) used it to estimate
emissions from tropical broadleaf raingreen trees. These errors did not result in large
differences in global emission rate estimates, but if they had we should not take it
as an indication that the models are advancing to an improved state where they are
diverging freely. That would only be the case if the models were diverging because
of some new understanding such as improved emission algorithms, emission factors
or approaches for upscaling emissions.
The statement that the lack of variability in BVOC emission estimates could
create the illusion of overly accurate BVOC emission estimates is a valid concern
although the tendency for the atmospheric chemistry modelling community to
modify BVOC emissions by 20–50 % (e.g., Houweling et al. 1998, Poisson et al.
2000; Ehhalt and Prather 2001), presumably because they are considered one of
the more uncertain parts of the model system, would seem to indicate otherwise.
In addition, there is a potential for model deviations to introduce unintended errors.
An example is the use of the Guenther et al. (1995) tropical forest emission factors
in models with different canopy environment models and foliar densities. The
410 A. Guenther

leaf-level emission factors recommended by Guenther et al. (1995) were based

on estimates of above-canopy concentrations, i.e., they represent the leaf-level
emissions required to give the observed above-canopy flux, assuming a certain
canopy environment model. Thus, these emission factors are only valid when used
with the Guenther et al. (1995) canopy environment model and foliar biomass. Even
for temperate and boreal regions, where the Guenther et al. (1995) emission factors
were based on leaf enclosure measurements, there was a potential confusion since
some models assumed that the emission factors were for sun leaves (Guenther et al.
1995), while others assumed a canopy average value (Pierce and Waldruff 1991).
To address this concern, Guenther et al. (2006) introduced a canopy-scale emission
factor so that, at least for standard environmental conditions, the emission rate
estimates of different models would be the same regardless of the canopy model
and LAI used. Disadvantages of this approach include obscuring the importance of
an accurate representation of canopy environment, which is needed for simulating
emission variations, and creating a false confidence in the accuracy of BVOC
emission models. A solution to this shortcoming is the reporting of both leaf- and
canopy-scale emission factors and a community effort to expand the observations,
including aircraft eddy covariance measurements, for assessing the accuracy of
BVOC emission models. Ideally these data would be available in a model testbed
that would streamline the process of testing and evaluating BVOC emission modules
against measurements over a wide range of spatial and temporal scales. This testbed
would consist of a suite of tools that allows extracting and comparing the predicted
BVOC fluxes or concentrations with a wide range of field measurements and/or
previous model predictions. The performance of new BVOC emission treatments
could then be quantified and compared to existing treatments before they are used
to assess the impacts of BVOC on regional air quality and the global Earth system.

Acknowledgements The National Center for Atmospheric Research is sponsored by the US

National Science Foundation.

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Chapter 15
Scaling Emissions from Agroforestry Plantations
and Urban Habitats

Susan M. Owen, C. Nicholas Hewitt, and Clare S. Rowland

Abstract Agroforestry plantations and urban habitats contribute importantly to

atmospheric volatile compound fluxes in densely populated areas. Simulation of
emissions from such habitats is associated with several key challenges, including
high spatial heterogeneity due to habitat fragmentation and high diversity of planted
tree species. On the other hand, plants in urban habitats and in agroforestry
plantations commonly receive more nutrients and water than species in natural
communities, resulting in higher production and potentially greater capacity for
volatile production per unit of land area. This chapter reviews the strategies for
simulation of biogenic volatile organic compound (BVOC) fluxes from urban
habitats and agroforestry plantations and provides an outline for parameterization
of volatile emission models for densely populated areas with high vegetation
fragmentation and large number of gardened, often exotic, tree species.

15.1 Introduction

In 2008, the global urban population exceeded the non-rural population for the first
time in history (United Nations 2008), and is currently more than seven billion
people. The increasing demands of a growing world population for food, fibre,
fuel, water, and shelter, is causing rapid land-use changes with global declines in

S.M. Owen ()

Biosphere Atmosphere Interactions, Centre for Ecology and Hydrology, Bush Estate, Penicuik,
Midlothian EH26 0QB, UK
e-mail: [email protected]
C.N. Hewitt
Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UK
C.S. Rowland
Monitoring and Observation Systems, Centre for Ecology and Hydrology, Library Avenue,
Bailrigg, Lancaster, LA1 4AP, UK

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 415
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 15,
© Springer ScienceCBusiness Media Dordrecht 2013
416 S.M. Owen et al.

natural forests and increases in agroforestry and urbanization (Foley et al. 2005).
It is predicted that by 2050, 70 % of the world’s population will live in towns or
cities (Seto and Shepherd 2009). The ecological and environmental impacts and
implications of rapid land-use change are well documented in the research literature
(e.g., Foley et al. 2005 and references cited therein). In this chapter, we focus on the
effects of these changes on fluxes of reactive biogenic volatile organic compounds
(BVOCs), which can have subsequent impacts on regional tropospheric chemical
reactions related to ozone and aerosol formation. Although the total urbanized
area is still relatively small, BVOC emissions from urban green areas significantly
contribute to the air quality of urbanized areas (Owen et al. 2003) with immediate
effects on human health. Furthermore, rapid expansion of urbanized environments,
agroforests and crop plantations implies that the effects of anthropogenic landscapes
to total emission budgets are expected to increase in the future. Urban environments
and agroforest have their specific environmental limitations that may significantly
differ from natural environments, and species compositions can also vastly differ.
Therefore, separate consideration of BVOC emissions from urbanized environments
and agroforests is warranted.

15.2 Importance of Urbanized Areas and Agroforests

for BVOC Emissions

15.2.1 Global Landcover of Urban Areas, Plantations

and Natural Forests

An overview of landcover of natural and plantation forests and urban land in the
major continents is shown in Table 15.1. Although the land footprint of cities
occupies less than 0.5 % of the Earth’s total land area (Schneider et al. 2009),
cities make a large contribution to the fluxes of reactive gases to the atmosphere.
Consequently, they are an important source term in the modelling of regional- to
global-scale biogeophysical processes such as atmospheric chemistry, redistribution
of atmospheric nitrogen oxides and aerosol production (Atkinson 2000). The urban
heat island effect influences local- to regional-scale climates (Quattrochi and Ridd
1994), and sensible and latent heat fluxes are modified by impervious surfaces
(Offerle et al. 2006), which probably affects precipitation regimes (Shepherd 2005).
Despite the growing importance of urban land area in regional- to global-scale
environmental issues, estimates of urban coverage vary with mapping method, and
the complexity and homogeneity of the urban land surface mosaics can propagate
large uncertainties (Schneider et al. 2009). Alvey (2006) states that “the urban forest,
which includes vegetation along urban streets and in urban parks, woodlots, aban-
doned sites, and residential areas, can comprise a significant percentage of a nation’s
tree canopy”, suggesting that this may amount to 25 % of mainland US total tree
canopy cover (Dwyer et al. 2000; Alvey 2006). As the result of rapid urbanization
worldwide, the importance of urban forests is expected to increase (Alvey 2006).
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 417

Table 15.1 Estimates of landcover (in 106 km2 ) for natural forests, forest plantations and urban
areas
Forest annual Planted Planted forest
Total forest change rate (%) forest area annual change rate Urban area
area (2010) (2000–2010) (2010) (%) (2000–2010) (2009)
Africa 6:74 0.49 0.154 C1.75 0.069a
Asia and Pacific 7:40 C0.19 1.20 C2.85 0.215
Europe 10:1 C0.07 0.693 C0.6 0.149
Latin America and 8:91 0.46 0.150 C3.23 0.095
Carribean
Near East 1:22 C0.07 0.151 C1.49
North America 6:79 C0.03 0.375 C2.46
World 40:3 0.13 2.64 C2.09 0.13
The data for forests and forest plantations are from FAO (2011), while the urban area data are from
Schneider et al. (2009) that provides MODIS satellite data at 500 m spatial resolution
a
Includes Near East

Biogenic volatiles can contribute significantly to total hydrocarbon flux from a

city. For example, Wang et al. (2010) reported that biogenic isoprene can contribute
more than 12 % to the non-methane hydrocarbon flux during August in Beijing.
Similarly, Saito et al. (2009) estimated that biogenic isoprene contributed about
40 % of the total non-methane hydrocarbon propylene-equivalent concentration
measured in summer in Nagoya, Japan. These biogenic sources of hydrocarbons in
a city can contribute in a significant way to the regional atmospheric chemistry. For
example, Harrison et al. (2006) estimated that biogenic isoprene removed around
16 % of the available OH in summer at an urban site in Birmingham, UK.
As human population and urbanization increase, so does the amount of land used
for agroforestry activities. The world’s total forest area is just over four billion
hectares (FAO 2010; Table 15.1), representing 31 % of total land area. Forest
plantations are being established at an increasing rate throughout much of the world,
and they now account for >6 % of global forest area (FAO 2011). There are huge
regional variations, with the UK having 68 % and New Zealand 24 % of forest
cover as plantation of exotic species. China has 16 % and France 13 % of forest
cover as plantation of native species (Brockerhoff et al. 2008). The species grown
in plantations are often fast-growing trees used for biofuel feedstocks and wood
pulp, and they are often high isoprene emitters (e.g., species of Populus, Salix, and
Eucalyptus; Harley et al. 1999).

15.2.2 Implications of Biodiversity for BVOC Emissions

from Urban, Plantation and Natural Forests

Agroforestry plantations and urban areas are considered to be species poor, although
this may not necessarily be the case (Brockerhoff et al. 2008). Ecosystem-sensitive
418 S.M. Owen et al.

management practices on plantations and greening of towns and cities may result
in higher biodiversity than expected, which at times can reach or even surpass the
biodiversity found in natural forests (Alvey 2006). Brockerhoff et al. (2008) report
that longer-rotation plantation forests, especially those managed with conservation
objectives, may differ little from managed natural forests (e.g., Keenan et al. 1997).
The type and amount of BVOCs synthesised and emitted are highly specific to
individual plant species, so the species composition of canopies within agroforestry
plantations and urban areas exerts a large influence on the type and amount of BVOC
flux from that canopy. Changes in plant species composition over a large region due
to new plantations or large-scale greening of urban space can therefore have far-
reaching impacts on the amount of BVOCs emitted and on the chemical reactivity
of these emissions, and hence have major impacts on tropospheric chemistry.
Since the emissions of BVOCs are species-specific, in order to construct a
“bottom-up” emission inventory, it is necessary to estimate the species composition
for a given habitat or canopy. In the case of an agroforestry plantation, the
composition will be dominated by the managed crop species. However, even in this
situation, understory or riverine species may make important contributions to the
total BVOC emissions, and hence should not be ignored.
In the case of the urban environment, the huge and rather unpredictable diversity
of tree species makes the integrated estimation of BVOC emission rates difficult.
Sun (1992) used the inverse of the Simpson’s diversity index (Simpson 1949) to
characterize the urban tree diversity:

N .N  1/
SDI D (15.1)
iP
Dk
ni .ni  1/
i D1

where ni is the number of individuals of species i, k is the total number of species in

the particular area, and N is the total number of individuals of all species together.
Hence, the greater the SDI the higher the species diversity. The SDI can be considered
as the “adjusted” number of species in a population based on species composition
(Sun 1992).
Sun (1992) reviewed the SDI of 21 towns and cities, principally in the US and
UK, and found that the SDI was less than 10 in 12 cases, and above 20 in one city
with a mean SDI of 9.5. This is very high diversity compared with tree diversity in
natural forests where the values of SDI are typically between 2 and 3 (Onaindia et al.
2004; Singh et al. 2005; Sharma et al. 2009), and may reach 8–10 in exceptionally
species-rich forests in biodiversity hotspots (Gimaret-Carpentier et al. 1998; Sharma
et al. 2009). Despite the high diversity, SDI may not be a useful metric when
assessing BVOC emissions, since BVOC emissions from individual tree species
vary enormously, and in any given urban area, may be dominated by very high
emission rates from a few individual specimens belonging to a few species. For
example, in Longyan County, Fujian, China, Sun records a total of 1,084 individual
trees (N) from 20 different tree species, with an SDI of 6. The most abundant six
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 419

species accounted for 941 individuals, with the remaining 143 individuals belonging
to 14 species. It is possible that these less common 13 % of individuals might
contain some very high BVOC emitting species, although this is impossible to
ascertain from currently published information as emission measurements have not
been made on most of these species.
Owen et al. (2003) used tree census data for different “urban morphology
types” (residential, industrial, transport, open space and commercial) together
with landcover maps to estimate the tree species present in the West Midlands
conurbation of the UK. However, this statistical representation of species diversity
was not tested by field surveys and may not include exotic species which might
make a relatively large contribution to overall BVOC emissions. Thus, choosing
how many, and which specific tree species to make measurements from is an integral
and necessary first step in any attempt to quantify BVOC emission rates from a
habitat, especially in the urban environment where the prevalence of exotics may be
high.
Nevertheless, the implications of the degree of species richness for BVOC
emissions if combined with information on vegetation coverage may still be
straightforward. A plantation of a high BVOC-emitting crop species, such as willow
(Salix spp.), oil palm (Elaeis guineensis) or poplar (Populus spp.), will result in a
canopy flux much greater than natural mixed forest. A highly built-up urban area
with few green spaces or plantings will have lower BVOC fluxes than the equivalent
areas of natural mixed forest, while an urban area rich with green spaces and gardens
might produce BVOC fluxes equivalent to or even exceeding some natural habitats
(Owen et al. 2003).

15.2.3 Exotic Species and BVOC Emissions in Urban

and Plantation Forests

An “exotic” species is a species growing outside its natural area of dispersal.

Richardson and Rejmánek (2011) performed an exhaustive literature survey, pre-
senting a “global list of invasive alien trees and shrubs and discussing taxonomic
biases, geographical patterns, modes of dispersal, reasons for introductions and key
issues regarding invasions of non-native woody plants around the world”. While
woody plants were not widely considered to be important invasive alien species until
fairly recently, Richardson and Rejmánek (2011) report that thousands of species of
trees and shrubs have been moved around the world, and that many have spread
from planting sites, and some are now among the most widespread and damaging
of invasive organisms. This is relevant because invasive exotics may emit more than
natives in the local flora (Llusià et al. 2010).
In an urban environment, it is doubtful whether the foliage and flowers of exotic
planted species are likely to alter the regional-scale BVOC flux, although they
may do so locally. Widespread garden and park plantings, and the escape and
420 S.M. Owen et al.

proliferation of large-flowered exotic species, might result in changes in regional

BVOC flux for the short periods of time when such a species flowers (e.g., Baghi
et al. 2012), but little work has been done to quantify these effects. For example,
the Rhododendron species are now widespread in the UK following introduction in
the nineteenth century, but to date, it is not known to what extent their foliage or
flowers contribute to total emissions of BVOCs. Large-scale monoculture plantings
of exotic species in agroforestry plantations have the same potential for contributing
to emissions of regional significance as their native counterparts, and the degree to
which a plantation species emits BVOCs with potential to cause detrimental changes
in air quality should be included in responsible decisions about the choices and
extent of agroforestry crops in any particular region.
The issue of exotics in urbanized areas is likely further gaining in relevance, es-
pecially in Northern Hemisphere higher latitudes where greatest rise in temperature
is predicted in the future with concomitant possibilities of many warmer climate
exotic species to be grown in urban areas (Niinemets and Peñuelas 2008).

15.2.4 Management Practices and Their Potential Effects

on BVOC Emission

Plants respond to biotic and abiotic stresses using a number of strategies, including
a change in BVOC production and emissions. The BVOC emission potential of
a non-stressed plant varies, depending on past environmental conditions, plant
physiological status, and phenology (Niinemets et al. 2011). Management practices
discussed in this section may cause physiological stress in plants for a period of
time. Other stresses associated with the urban and agroforestry environments, but
not a direct result of management strategies, are discussed in the next section. The
directions and magnitude of the BVOC response to any stress depends on plant
species, the health of the plant before the onset of stress, and on the severity and
duration of the stress, and the potential for synergies among several coincident stress
factors (Niinemets et al. 2010a).
Management practices in urban green spaces and agroforestry plantations may
include application of inorganic or organic fertilizers, irrigation, and cropping,
felling, pruning or mowing. These practices may be responsible for additional
emissions, and also restrict the vegetation, e.g., managed to an age limit. The effects
that these practices have on BVOC emissions are likely to be plant species specific,
and are likely to be different for isoprene and other compounds emitted immediately
following synthesis, and those compounds emitted by vapourization from stored
pools within the plant tissues (Grote et al. 2013). Some likely responses of BVOC
emissions to different management practices are summarised in Table 15.2.
Fertilizers appear to have different effects on BVOC emissions. Results vary
with plant species, amount of fertilizer applied and the pre-fertilized status of the
growing medium. For example, Blanch et al. (2007) found that fertilizer treatments
Table 15.2 Review of responses of plant BVOC emissions in response to management and other stresses
Response (Study)
Management Isoprene/ Monoterpenes
practice/ instantaneously from stored Oxygenated
Stress emitted terpenes tissue pools Sesquiterpenes compounds
Fertilizer "(4, 5); no change (6) #(1) "(2, 4) "(3, 4) "(13)d
Irrigation "(21) "(21) "(27); # (27)c "(25); no change (25)b
Cropping, felling, pruning or mowing "(26) "(9) "28 "(7, 8)
Plant age "(12) "(10, 11) "(33)c ; no change (32) #(34); "(34)b,c
Drought/desiccation stress No change (14); #(20 23, 31); " 31a No change (31); #(24) No change (25)b ; #(25)
#(15, 16, 17, 18,
19, 22)
Herbivory stress in plantations " short-term (35); "(29, 30) "(29, 30) "(29)
# long-term (35)
Overcrowding/shading #(37) No change (39)b,e ; #(38, 39) #(40) # (41)
Studies: (1) Blanch et al. (2007); (2) Blanch et al. (2012); (3) Rinnan et al. (2011); (4) Ormeño et al. (2009); (5) Possell et al. (2004); (6) Funk et al. (2006);
(7) Seco et al. (2007); (8) Davison et al. (2008); (9) Räisänen et al. (2008); (10) Kim et al. (2005); (11) Street et al. (1997a); (12) Street et al. (1997a); (13)
Hörtnagl et al. (2011); (14) Steinbrecher et al. 1997; (15) Tingey et al. 1981; (16) Sharkey and Loreto (1993); (17) Fang et al. (1996); (18) Lerdau et al. (1997);
(19) Brilli et al. (2007); (20) Lavoir et al. (2009); (21) Peñuelas et al. (2009); (22) Pegoraro et al. (2004); (23) Bertin and Staudt (1996); (24) Ormeño et al.
(2007); (25) Filella et al. (2009); (26) Brilli et al. (2011); (27) Llusià and Peñuelas (1998); (28) Piesik et al. (2011); (29) Schaub et al. (2010); (30) Staudt
and Lhoutellier (2007); (31) Staudt et al. (2008); (32) Agelopoulos et al. (2000); (33) Hakola et al. (2001); (34) Bracho-Nunez et al. (2011); (35) Loreto et al.
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats

(2006); (36) Brilli et al. (2009); (37) Harley et al. (1996); (38) Owen et al. (2002); (39) Tarvainen et al. (2005); (40) Staudt and Lhoutellier (2011); (41) Folkers
et al. (2008)
a
Depends on severity of stress
b
Depends on compound
c
Depends on plant species
d
Soil with vegetation
e
Depends on the site of emission
421
422 S.M. Owen et al.

reduced the monoterpene emissions from Pinus halepensis by 40 %, yet Blanch

et al. (2012) found five-fold increased monoterpene emissions from phosphorus-
stressed seedlings of Pinus pinaster. Both of these species emit monoterpenes from
tissue pools, and both experiments were conducted on tree seedlings growing in
pots. Rinnan et al. (2011) found a stimulation of the sesquiterpene “-selinene from
Salix phylicifolia growing in field plots, after administering annual additions of
NPK fertilizer. This was contrary to their expectations that increased soil nutrient
availability would decrease BVOC emissions, assuming an increased allocation of
carbon to growth (Bryant et al. 1983; Sorensen et al. 2008). Ormeño et al. (2009)
reported that mono- and sesquiterpene basal emissions from Rosmarinus officinalis
(a terpene-storing species) and Quercus coccifera (a non-storing species) increased
with a moderate application of organic fertilizer at 50 t ha1 , which the authors
proposed were optimal N conditions. Possell et al. (2004) also found that increasing
nutrient availability increased isoprene emissions from Quercus robur growing in
pots. Analogous increases have been observed in velvet bean (Mucuna sp.) by
Harley et al. (1994) and in white oak (Quercus alba) by Litvak et al. (1996). Yet
Funk et al. (2006) found that leaf-level emission at a given canopy height did not
differ between fertilized and unfertilized 6-year-old Eucalyptus saligna forest trees.
They proposed that this was linked to the leaf nitrogen content, which also did not
vary with fertilizer treatment.
The emission responses to fertilizer treatment at ecosystem scale are even more
difficult to predict. For example, Hörtnagl et al. (2011) reported that application
of organic fertilizer significantly increased methanol emissions from a grassland.
However, these emissions probably reflected enhanced microbial activity in the
soil associated with the applied manure (Hörtnagl et al. 2011). A meta-analysis
of published results is needed to attempt to tease out the underlying cause and
effects, and more manipulation experiments are needed to understand better the role
of nutrients, and hence fertilizer treatment, on BVOC emissions not only from the
plant tissues, but also from the soil substrate.
Cropping, felling, pruning, mowing etc. can result in a burst of the emissions of a
wide range of BVOCs, particularly the oxygenated compounds. In a comprehensive
overview of short-chain oxygenated volatiles, Seco et al. (2007) reviewed emissions
of formic and acetic acids, acetone, formaldehyde, acetaldehyde, methanol, and
ethanol, some of which have been observed as a response to cutting, harvesting or
mowing (e.g., Davison et al. 2008; Fall et al. 1999, 2001). Hörtnagl et al. (2011) also
reported high emissions of methanol during and after cutting a grassland meadow,
which they attributed to the wounding of the plant material and subsequent depletion
of the leaf internal aqueous methanol pools. Räisänen et al. (2008) measured higher
atmospheric concentrations of monoterpenes at felled sites compared to non-felled
sites within a Scots pine (Pinus sylvestris) plantation, and considered that the
logging residue was the most important factor explaining the elevated monoterpene
concentrations. These practices are carried out regularly on agroforestry plantations
and within many different urban green spaces. Episodes of maintenance practices
will contribute significantly to the BVOC flux from these canopies.
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 423

BVOC emissions from agroforestry plantations or urban green spaces may also
be modified if these spaces are managed to constrain the age of the vegetation
growing there. Niinemets et al. (2011), reviewing a number of studies about the
factors affecting BVOC emission potentials, report that age (leaf age, plant age)
is one of the factors that can alter species-specific emission potential values by
more than an order of magnitude. An emission potential is the emission rate of a
particular compound at a standard set of environmental conditions, usually 30 ı C
and 1,000 mol m2 s1 light. Guenther et al. (2006) added further parameters to
the definition of standard conditions, including leaf age and soil water conditions.
The direction in which the age of a plant alters BVOC emission potential is
species-specific, and it may also depend on environmental and biotic conditions.
For example, Kim et al. (2005) found that total emission rates and speciation of
monoterpene compounds varied significantly with tree species, age, and season. In
their study, total emissions from Cryptomeria japonica and Pinus koraiensis were
higher for older trees than for younger trees. Yet, significantly higher emissions
were found from younger trees for Chamaecyparis obtusa (Kim et al. 2005). Street
et al. (1997a) found that BVOC emission potential of mature Pinus pinea forest trees
was twice that of young plantation trees growing nearby in similar environmental
conditions (P < 0.05). Wiberley et al. (2005) found that in kudzu (Pueraria lobata),
the age dependency of the capacity to emit isoprene was in turn controlled by
environmental growth conditions.
Taking account of leaf age when extrapolating BVOC emissions from leaf- and
branch-scale to canopy-scale flux is possible if the species composition and age
structure of the canopy is known, and if investigations have already been made on
the effect of leaf age on BVOC emissions to provide the species-specific functional
relationships. Simulation of changes in isoprene emissions due to age can be made
using algorithms which can be found embedded in e.g., the Model of Emissions
of Gases and Aerosols from Nature (MEGAN) for global isoprene flux emissions
(Guenther et al. 2006, 2012; Guenther 2013). Leaf age is also indirectly embedded
in some of the seasonality functions used to characterize seasonal variations in
emissions (Grote et al. 2013; Niinemets et al. 2013). These approaches (species-
specific measurements and simulation algorithms) are likely to be feasible for
monoculture agroforestry plantations, but they can be complex to apply for a highly
fragmented urban green-space canopy.

15.2.5 Biotic and Abiotic Stresses and Their Potential Effects

on BVOC Emissions

Because most plants are sessile, they need to be able to cope with many changes
in their environment, such as light intensity, temperature, water availability and
other abiotic factors, as well as biotic factors such as herbivory and competition.
When these factors shift out of the normal range, due to management strategies
in plantations for example, then plants experience stress that can result in slower
424 S.M. Owen et al.

growth rate, impaired reproduction and even death. Environmental and biotic stress
can also select for shifts in ecological traits (both short-term and long-term),
with potential impacts on biological diversity, ecosystem functioning and carbon
sequestration (Vickers et al. 2009). Urban vegetation and trees in particular are
likely to experience several different types of stress. For example, street trees may
be growing in concrete and paved surfaces where they may be exposed to drought
stress, which has different effects on BVOC emissions depending on tree species,
type of BVOC emitted, severity and duration of stress, and general conditions of the
tree. This is comprehensively reviewed in Laothawornkitkul et al. (2009) and Possell
and Loreto (2013). A review of multiple stresses on BVOC emissions is provided
by Holopainen and Gershenzon (2010). Urban vegetation may be nutrient stressed
because their fallen leaves are swept away and are not allowed to decompose in situ
and recycle soil nutrients. They may be exposed to high levels of pollutants, and
may even be used as natural pollutant filters alongside roads. Trees and other plants
growing in towns and cities are also vulnerable to mechanical damage caused by
heavy management strategies and vandalism.
In contrast, trees and other plants growing in agroforestry plantations and
gardens are more likely to be protected against stress, as productivity depends upon
good growing conditions. Agroforestry managers will aim for optimum fertilizer
application and watering regimes. However, planting closer than the prescribed
optimum can result in competition for resources, therefore trees and plants in badly
managed plantations may be stressed. In most cases, prolonged moderate stress and
strong rapid stress result in reduction of the constitutive BVOC emissions for all
stresses studied so far (Niinemets 2010; Fineschi and Loreto 2012). Thus, an urban
green-space canopy or a plantation canopy suffering any of a range of biotic and
abiotic stresses will result a change in magnitude and chemical speciation of the
BVOC flux from that canopy. Some effects on BVOC emissions from different
stresses likely to be experienced by plants growing in an urban environment and in
agroforestry plantations are summarised in Table 15.2 and we also refer to chapters
from Calfapietra et al. (2013), Grote et al. (2013) and Possell and Loreto (2013) in
this book.

15.3 Estimating BVOC Emission Rates from Urban

and Plantation Canopies

An overview of BVOC flux estimates for agroforestry plantation species and

urban canopies is presented in Table 15.3. In this section, we discuss some
methods, complexities and uncertainties associated with these emission estimates.
For monocultures and for canopies with very few species of uniform age, simple
extrapolations still can sometimes produce estimates that are comparable with
canopy-scale measurements (Guenther 2013). Advanced canopy parameterizations
account for light extinction and changes in emission potential through the canopy,
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 425

Table 15.3 BVOC canopy-, branch- and leaf-level emission fluxes reported from agroforestry
plantations and urban canopies
15.3a Canopy BVOC emission estimates (mg C m2 h1 ) for dominant species
Dominant species Flux BVOC Notes Reference
a b
Acacia nilotica 0.02 Isoprene , , Lext, aa Harley et al. (2003)
a b
Acacia nigrescens 2.7 Isoprene , , Lext, aa Harley et al. (2003)
Elaeis guineensis 0.36 Estragole E, 24m Misztal et al. (2010)
Elaeis guineensis 26.5 Isoprene E, md Misztal et al. (2011)
Hevea brasiliensis 0.15–1 Monoterpenes E Baker et al. (2005)
Picea abies 0.65–1.08 Monoterpenes Cmod, md Forkel et al. (2006)
Pinus sylvestris 0.15 Monoterpenes E, ms Räisänen et al. (2009)
b
Pinus sylvestris 0.92 Monoterpenes ,E Räisänen et al. (2009)
b
Salix viminalis 3.1 Isoprene ,C Olofsson et al. (2005)

15.3b Leaf- or branch-level BVOC emission estimatesc

Species Flux Notes Reference
Isoprene (g C m2 h1 )
b
Acacia nigrescens 11–86 ,L Possell and Hewitt (2011)
b f g
Eucalyptus globulus 560–3,760 , , ,L Guenther et al. (1991)
b g
Eucalyptus globulus 10,800 , ,L Monson et al. (1991)
b
Eucalyptus globulus 3,530–4,410 ,L Street et al. (1997b)
b g
Eucalyptus globulus 6,100 , ,L He et al. (2000)
Eucalyptus globulus 440–1,300 L Winters et al. (2009)
Eucalyptus saligna 5,400–6,700 L Funk et al. (2006)
b
Picea abies 74 ,L Forkel et al. (2006)
b
Populus trichocarpa 9,720 ,L Guidolotti et al. (2011)
b
Populus deltoides 5,400 ,L Guidolotti et al. (2011)
Populus  euramericana 5,400–6,050 b
,L Guidolotti et al. (2011)
b
Populus nigra 3,900–8,200 ,L Guidolotti et al. (2011)
b
Populus deltoides x Populus trichocarpa 2,590 ,L Ryan et al. (2009)
b
Quercus serrata 6,050 ,L Tani and Kawawata (2008)
b
Quercus mongolica 6,050 ,L Tani and Kawawata (2008)
b
Quercus dentata 6,480 ,L Tani and Kawawata (2008)
b
Quercus aliena 3,900 ,L Tani and Kawawata (2008)
b
Quercus robur 650–3,880 ,L Lehning et al. (2001)
Isoprene (g C g1 h1 )
d,i d i
Morus alba 1.8–53 , ,L Singh et al. (2007)
d d
Picea abies 0.01–0.07 ,L Filella et al. (2007)
Picea abies 0.6 L, md Grabmer et al. (2006)
b,h b h
Picea abies 0.32–1.7 , ,L Grabmer et al. (2006)
b b
Picea mariana 6.7 ,L Fulton et al. (1998)
b b
Picea sitchensis 11.5 ,L Hayward et al. (2004)
Picea sitchensis 0.004–1.3 L Street et al. (1996)
b b
Populus tremula 45 ,L Hakola et al. (1998)
b b
Nandina domestica 17.5–22 ,L Winer et al. (1983)j
(continued)
426 S.M. Owen et al.

Table 15.3 (continued)

Species Flux Notes Reference
b b
Robinia pseudoacacia 118–192 ,L Geron et al. (2001)
b b
Salix phylicifolia 28 ,L Hakola et al. (1998)
Monoterpenes (g C m2 h1 )
b e f g
Eucalyptus globulus 475–6,400 , , , ,L Guenther et al. (1991)g
b
Eucalyptus globulus 175–530 ,L Street et al. (1997b)
b
Eucalyptus globulus 2,920–3,320 ,L Winters et al. (2009)
b
Picea abies 300 ,L Forkel et al. (2006)
b
Quercus coccifera 2,350 ,L Staudt and Lhoutellier (2011)
Monoterpenes (g C g1 h1 )
b
Cupressus forbesii 1.5 ,L Arey et al. (1995)
b
Hevea brasiliensis 1.8–83 ,L Wang et al. (2007)
b
Larix sibirica 4.6–15.4 ,L Ruuskanen et al. (2007)
d
Picea abies 0.09–0.9 ,L Filella et al. (2007)
Picea abies 1.8 L, md Grabmer et al. (2006)
b
Picea abies 0.5 ,L Grabmer et al. (2006)
b
Picea mariana 2.4 ,L Fulton et al. (1998)
b
Picea sitchensis 2.6 ,L Hayward et al. (2004)
Picea sitchensis 0.01–1.9 L Street et al. (1996)
Pinus sylvestris 0.05–3.3 L Komenda and Koppmann (2002)
Pinus halepensis 27.8 L Blanch et al. (2007)
b
Populus tremula 4.1 ,L Hakola et al. (1998)
b
Salix phylicifolia 0.3 ,L Hakola et al. (1998)

15.3c Leaf- or branch-level BVOC emission estimatesc

Flux or
Location concentration Notes Reference
Isoprene (mg C m2 h1 )
Frankfurter Stadtwald, Germany 0.75 C Steinbrecher et al. (2000)
Houston, USA up to 0.0.6 C Park et al. (2011)
London, UK 0.11 C, ma Langford et al. (2010)
b
Las Vegas, USA 0.14–0.21 , Lext Papiez et al. (2009)
West Midlands, UK 0.04–1.46 Lext Owen et al. (2003)
Greater London, UK 1 Lext MacKenzie et al. (1991)
Tucson, Arizona, USAk 2.5 Lext Diem and Comrie (2000)
Isoprene (ppbv)
Changping district, Beijing, China 0.7 C Wang et al. (2010)
Nagoya, Japan 0.7 C Saito et al. (2009)
28 cities in the US 0.05–2.5 C Baker et al. (2008)
Bilbao, Spain 0.12 C Durana et al. (2006)
Birmingham, UK 0.2 C Harrison et al. (2006)
43 cities in China 0.02–0.8 C Barletta et al. (2005)
(continued)
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 427

Table 15.3 (continued)

Flux or
Location concentration Notes Reference
Monoterpenes (mg C m2 h1 )
Frankfurter Stadtwald, Germany 0.02 C Steinbrecher et al. (2000)
b
Las Vegas, USA 0.09–0.4 , Lext Papiez et al. (2009)
West Midlands, UK 0.02–0.09 Lext Owen et al. (2003)
Tucson, Arizona, USAk 0.3 Lext Diem and Comrie (2000)
Notes key
a
Assuming 100 % canopy cover for the species
b
Standardised to 30 ı C (and 1,000 mol m2 s1 light for light-dependent emissions)
c
Emission rates expressed per mass of leaf material cannot be converted to leaf area based emission
rates without specific measurements of leaf mass per unit area
d
At different temperatures
e
Depending on compound
f
Depending on leaf age
g
Cited in Winters et al. 2009
h
Depending on model used
i
Field data in different seasons
j
Cited in http://bai.acd.ucar.edu/Data/BVOC/index.shtml
k
Urban vegetated residential area
24m – 24 h mean of measured flux, md – mean midday measured flux, ms – mean summer
measured flux, ma – mean autumn measured flux, aa – area-averaged, C – canopy, Cmod –
modelled canopy; E – ecosystem, L – leaf/branch, Lext – leaf measurements extrapolated to
canopy-scale flux

as well as changes in physiology and phenology of the vegetation (Guenther 2013).

However, there remain substantial uncertainties associated with extrapolating leaf
emission measurements to whole canopy-scale flux which are summarised by Guen-
ther et al. (2006): “(1) a limited understanding of chemical sinks and deposition
losses within vegetation canopies (2) artificially disturbed emission rates due to
the enclosure, (3) differences between the functioning of individual ecosystem
components (e.g., leaves) and the entire ecosystem, and (4) limited sample size
within the enclosure (relative to the whole landscape), as well as uncertainties
associated with canopy microclimate models themselves.” Furthermore, a specific
limitation of available BVOC models in agroforest plantations is the high degree of
foliage clumping in many fast-growing agroforest species, implying that accurate
parameterization of radiative transfer in such canopies would require more complex
models (Cescatti and Niinemets 2004). Ideally a combination of bottom-up and
top-down approaches to quantifying canopy flux is likely to give the most accurate
estimates of BVOC flux and its speciation from an urban or agroforestry plantation
canopy.
In general, two different approaches are possible for estimating emissions from
plantations or urban habitats. The first method is to construct a “bottom-up” emis-
sions inventory, analogous to that used to estimate emissions from anthropogenic
pollutant sources. Until now, this method is by far the most widely used for
constructing BVOC inventories. Briefly, it requires the identification of individual
428 S.M. Owen et al.

emission sources (i.e., which tree species emit which compounds), an emission
factor for each tree species and each BVOC species at standard environmental
conditions, per unit leaf area or unit biomass, an “activity” factor (i.e., the amount
of leaf area or biomass present), and finally a mathematical description of how
temperature, light intensity and all other external factors moderate the “standard”
emission rates. These are then combined to give a “canopy-scale” emission rate,
which may be moderated by “in-canopy” losses due to chemical reactions or
deposition. The “bottom-up” approach with its various ramifications is discussed
further in the next section. The second, alternative, method is to use a direct “top-
down” canopy-scale measurement of the integrated emission rate from the habitat
using one of the several flux measuring methods that are currently being developed,
based on an understanding of boundary-layer micrometeorology. Indirect ‘top-
down’ approaches using atmospheric concentrations to infer emission rates by
inverse modelling are also possible, utilizing measurement data from aircraft (e.g.,
NASA’s Global Hawk aircraft) or satellites (e.g., GOSAT, SCIAMACHY or OMI)
(Ashworth et al. 2013 for a discussion on satellite-based estimates).

15.3.1 “Bottom-Up” Approach

A description of the “bottom-up” approach is given by Keenan et al. (2009b), and

summarised here in the most simplified form as:

i Dk
X
Fc D Epsi B si fe .T; Q; : : :/; (15.2)
i D1

where Fc is the canopy flux, Epsi is the species-specific emission potential (emission
factor) per unit dry mass, Bsi is species biomass and fe (T,Q, : : : ) is a function
describing the effects of environmental drivers on the emission rate. This function
routinely includes instantaneous temperature and light effects, but it may also
include the effects of CO2 concentration (Wilkinson et al. 2009), drought, leaf
age and other factors depending on the model parameterization (Guenther et al.
2006; Grote et al. 2013). The function fe (T,Q, : : : ) can be a composite of best-
fit environmental dependencies (Guenther et al. 2006), or link the emissions to
photosynthetic light (Niinemets et al. 2002) or dark reactions (Martin et al. 2000).
The emission model is typically driven by incident light and air temperature, and
can consider the canopy as a big leaf or a two-big-leaf with sunlit and shaded
leaf area fractions (Dai et al. 2004; Guenther et al. 2006; Keenan et al. 2009a).
Models with layered canopy have also been used simulating the vertical variations
in temperature, light and Epsi (Guenther et al. 2006; Guenther 2013; Niinemets et al.
2013) However, the current key limitation of these models is the lack of information
of species-specific variations in emission potentials within the canopy (Niinemets
et al. 2010b).
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 429

Because these models are driven by light, temperature, and may include water
availability and other key environmental drivers, it is potentially possible to run
these models under different climate change scenarios (Keenan et al. 2009a;
Schurgers et al. 2009; Young et al. 2009). It is also possible to perturb the vegetation
data to simulate land-use changes, thereby predicting possible changes in BVOC
emissions resulting from future climate, planning and management changes.
Estimating correct leaf-level emission potentials for the component species of a
canopy is of paramount importance for parameterizing these models. In the urban
environment, trees from a large number of species are present, including both native
and exotic species, as individual specimens (for example in domestic gardens or
common areas), as linear plantings along streets, as groups in squares or other
smaller open spaces, or in larger numbers in parks or other large open spaces. In
size, they vary from small shrubs and saplings in gardens or as new plantings, to
fully-grown specimens which may reach 40 m or more in height in large gardens or
parks. The number of different species present in an urban area varies with land use
with suburban residential areas typically containing many more exotic species than,
for example, dense inner-city areas. Agroforestry plantations typically contain far
fewer species. In both urban and plantation canopies, the extrapolation process also
requires knowledge of the contribution in terms of biomass and leaf area for each
component species.

15.3.2 Estimating Urban and Plantation Canopy Species

Composition and Biomass by Remote Sensing

Remote sensing is the remote measurement of reflected radiation, by a sensor, often

for a range of different wavelengths, and it can provide data on vegetation cover and
type for the ‘bottom-up’ approach to estimating canopy flux of BVOCs. Remote
sensing datasets vary in:
Type of sensor e.g., optical, radar or Light Distance And Ranging (LiDAR).
Spectral resolution – number and location of wavelengths that are sampled.
Spatial resolution – size of the area on the ground represented by a pixel in an image.
Radiometric resolution – precision with which radiation is observed.
Temporal resolution – frequency with which repeated images of a site are acquired.
Consequently, the information that can be derived from the remote sensing data
varies considerably depending on sensor specifications and mode of sampling.
A range of techniques have been explored for characterizing urban greenspace and
the urban canopy from remote sensing data. The techniques can be grouped in
several ways, but for the purposes of this chapter, they are grouped according to
spatial resolution:
High resolution – pixel size <5 m (often <1 m), includes data from LiDAR, aerial
photography and high-resolution satellite data from satellites such as IKONOS,
430 S.M. Owen et al.

Quickbird and Worldview. High-resolution sensors are typically designed and

applied to small test sites, where high spatial detail is required for a single-point
in time.
Medium resolution – pixel sizes of roughly 10–30 m. They are principally designed
to meet regional mapping requirements, including mapping change over time.
Coarse resolution – defined here as pixels >250 m that are suitable for large-scale
or global mapping and suitable for monitoring change over time.
In addition to these methods, tools such as Google Earth and Google Streetview
increase the level of information about species and management that can be gained
remotely. However, the data are limited both in temporal coverage and, in the case
of Streetview, its spatial coverage, but it may provide a useful supplement to field
data in some cases.
There are potential synergies between different datasets, so, for example, com-
bining LiDAR, aerial photography and field measurements enables the delineation
of tree crowns and identification of species and height estimation. Clearly, the
best approach is a combination of remote sensing to provide very high resolution
raster and landcover data, with classification and survey work for ground-truth, and
for obtaining plant-level measurements and observations which are important for
BVOC emission estimates.

15.3.2.1 LiDAR

LiDAR is a high-resolution technique that uses laser pulses to measure the distance
from the sensor to the ground. Post-processing of the data then enables construction
of a digital surface model, which maps the height of features, such as buildings,
vegetation and the ground. When the laser pulse reaches a vegetation canopy, it
will typically penetrate some distance into the canopy before being reflected back
to the sensor. Consequently, LiDAR is likely to underestimate the true height of
the canopy, unless a calibration or correction is applied (Gaveau and Hill 2003;
Anderson et al. 2006). LiDAR can be segmented to automatically delineate tree
crowns (Shrestha and Wynne 2012). Segmentation is the use of an algorithm to
identify homogeneous areas, in this case corresponding to tree crowns. Once tree
crowns are identified as discrete objects, then object-based processing methods
enable other attributes derived from remote sensing datasets or field observations
to be attached, such as species, tree height and crown diameter. The identification of
individual trees is especially useful in urban areas, as species, age and management
may be highly variable over small areas (Ardila et al. 2012). Once species are known
from aerial photography or field data then relationships between the LiDAR data and
tree height, crown diameter and biomass can potentially be developed for the main
tree species, if field calibration data are available (Shrestha and Wynne 2012).
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 431

15.3.2.2 High-Resolution Datasets

High-resolution datasets are currently provided by three sources, aerial photos, high-
resolution aircraft measurements such as those taken with AVIRIS (hyperspectral
imaging) and high-resolution satellite data, such as Quickbird and IKONOS. Aerial
photographs are able to provide higher resolution images than the satellite sensors.
High-resolution aircraft and satellite data can be processed in the same manner as
aerial photographs or medium-resolution satellite data. Manual interpretation, by an
expert, of aerial photographs can yield information about a range of urban canopy
parameters including location and size of tree canopies, tree species in very fine-
resolution images (Valerie and Marie-Pierre 2006) and, with stereo-photography,
estimates of tree height (St-Onge et al. 2004). Identification of features in aerial
photos is based on interpreting subtle differences in shape, texture, size, colour,
shadow and spatial context (Morgan et al. 2010). It is often difficult to apply
automatic processing methods to aerial photographs, because of the limited spectral
information, whereas expert manual interpretation may produce better results, but
can be time-consuming. High-resolution hyperspectral aircraft data can compensate
for these limitations yielding extreme level of detailed information, albeit automatic
data processing is still somewhat difficult due to lack of generalized algorithms
(Somers and Asner 2012).

15.3.2.3 Medium-Resolution Satellites

Medium-resolution satellite sensors are valuable for mapping urban areas and
greenspace in urban areas, because a single image is able to cover an entire city
or conurbation. For example, Landsat images are approximately 180 km  170 km.
This enables large areas to be mapped relatively easily. There are two main methods
for classifying urban landcover, either image classification where each pixel is
assigned a landcover type based on its spectral characteristics, or spectral mixture
analysis (SMA) methods which assign a percentage of different landcover types to
a pixel. The key limitation of medium-resolution satellite data for urban areas is
that their pixel size is poorly suited to the highly heterogeneous urban landscape.
Individual pixels will usually contain a complex mix of buildings, roads, shadows,
trees and grass. Spectral mixture analysis methods are often preferred over image
classification, as they offer a more flexible method for dealing with mixed pixels.
The number of sub-pixel components that can be estimated using SMA is limited by
the spectral resolution of the sensor, and so typically three components are used for
urban mapping: green vegetation, substrate and dark surface (Small and Lu 2006).
However, the SMA results can be augmented by image classification results, where
more classes are typically resolvable, including coniferous, deciduous and grass
classes in parks or densely vegetated suburban areas.
432 S.M. Owen et al.

15.3.2.4 Coarse-Resolution Satellites

There are two main advantages of coarse-resolution satellite data. First, the data
are typically free, although there may be licensing restrictions and costs for certain
products or data delivery charges in some cases. Second, the temporal coverage is
high, meaning that change over short time periods can potentially be monitored.
Variation in vegetation indices derived from coarse-resolution data have been
shown to track increasing urbanization (Dallimer et al. 2011), and can be used to
characterize changes in vegetation seasonality (Guenther 2013).
The availability and cost of the different types of data varies widely and is
an important consideration. Most remote sensing datasets have to be purchased
from satellite operators or resellers of aerial photo archives, with the exceptions
being data from coarse-resolution sensors and data from the Landsat series of
sensors. The availability of archived datasets differ widely across sensors, with
aerial photographs providing a long, but often sparse, time-series extending over
60 years in some cases. Conversely, satellite archives cover shorter time periods, but
have many more acquisitions per site.

15.3.2.5 Use of High-Resolution Remote Sensing Data to Identify

and Quantify Agroforest Canopies

The “bottom-up” approach to mapping tree species and modelling emissions is

also appropriate for agroforestry canopies. Remote sensing of agroforestry is
typically simpler as the relatively large, homogeneous, monospecific, even-aged
stands are more suited to the current capabilities of satellite sensors, than the
heterogeneity of the urban environment. The techniques for agroforestry include
the methods discussed in the section on remote sensing of the urban canopy, but
also include radar methods. Radar is sensitive to the structure of the woodland and
Interferometric radar (InSAR) is particularly useful for estimating canopy height
or biomass (Balzter et al. 2007), especially if a good quality digital terrain model
(DTM) exists or can be derived from the InSAR data.

15.3.3 Urban Land Classification and Ground Survey

An alternative or supplementary approach to remote sensing are ground surveys of

the vegetation. This approach requires at least 1 km2 resolution raster data of land-
and vegetation-cover characteristics (for example %woodland, % roads, %suburban,
%dense urban, %inland water, etc.), but provides a wealth of ground-truthed data.
Briefly, the land- and vegetation-cover characteristics for each pixel (e.g., km2 )
in the raster dataset for the area of interest are analysed using PCA and cluster
analysis to generate a classification of the urban landscape. This classification is
then used for a stratified sampling of vegetation. Depending on available resources,
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 433

sample plots are randomly allocated within each characteristic cover-type of the
classification strata, and weighted to the number of pixels in each class. Details
are recorded, such as species, age, height, diameter-at-breast-height, crown spread,
LAI, and leaf dry mass per unit area (LMA). The health/stress status is also noted
as this is likely to affect BVOC emissions. It is also worth recording any obvious
management practice or damage, such as branch trimming, coppicing, damaged
bark. When the tree inventory of the sample plots is complete, the data can then
be extrapolated to the sampled area or forest footprint using the knowledge of
total coverage for each land- and vegetation-cover characteristic contributing to that
square. The classification of each sampled square then enables simple extrapolation
to the whole urban landscape. It is clear that a very high uncertainty is associated
with this extrapolation method. However, the classification should embrace all the
major land/vegetation cover types within the area of interest. It is also likely that
local authorities will tend to use a fairly limited range of tree species for their
planting programmes, and that individuals will tend to select plants for their gardens
which local garden centres supply, and will also select plants that they admire in
their neighbours’ gardens. Thus, there may be a natural restriction on the extent of
vegetation diversity within a neighbourhood, and a correspondingly lower degree of
uncertainty in bottom-up extrapolations of urban vegetation.

15.3.4 Measuring Leaf- and Branch-Level Emission Rates

Once the canopy composition is determined, species-specific emission potentials

can be determined either from literature surveys (e.g., Stewart et al. 2003; Keenan
et al. 2009b) or by measurements specific for the canopy (e.g., Owen et al. 2003).
Obtaining representative, meaningful and reliable data on the emission rates of
BVOCs from trees, whether in their natural habitat, in the urban environment, or in
a managed agroforestry plantation, poses both conceptual and technical challenges
(Hewitt et al. 2011). These challenges arise because (1) different tree species emit
different blends of BVOCs, each at different rates, (2) at the leaf level, emission
rates vary with leaf age, leaf temperature, soil moisture and for some BVOCs with
light intensity, (3) at the tree scale, size of tree (biomass or leaf area) influences
BVOC emission rates, (4) BVOC emission potentials for an individual plant can
change according to the conditions of light, temperature, stress in the days or weeks
preceding the measurements, and (5) as discussed above, BVOC emission rates may
be influenced (positively or negatively) by biotic and abiotic stresses. These issues
are discussed in depth by Niinemets et al. (2010b, c).
There is already a large database of BVOC emission potentials for a range
of plant species throughout the world (http://bai.acd.ucar.edu/Data/BVOC/index.
shtml), in addition to a large source of research literature, but the existing resources
must be used with caution. As well as the challenges listed above, sampling and
analytical techniques can also be a source of uncertainty (Niinemets et al. 2011).
Niinemets et al. (2011) propose standardization of experimental and calculation
434 S.M. Owen et al.

protocols as well as critical examination of past reports to develop accurate emission

potential databases in the future. In view of this, it is therefore better if BVOC
emission measurements are made according to this protocol, from a sample of plant
species within the area of interest. It would be even better if extended measurements
can be made over 24 h, and during different portions of the growing season.
Alongside the excellent overview of Niinemets et al. (2011), Ortega and Helmig
(2008) and Ortega et al. (2008) also review enclosure methods of sampling and
measuring BVOC emission rates. Here, we provide a brief summary of methodology
and direct readers to these reviews for further details.
Estimates of leaf-level BVOC emission rates may be obtained by measuring the
concentrations of emitted compounds in the head-space above leaf tissue, either in
a static system without air flow (e.g., Hewitt and Street 1992), or in a dynamic
system where air flows over the leaf and through the enclosure (e.g., Hayward
et al. 2004). In static systems, it is possible that a compensation point is reached,
as the concentration builds up over time, such that emission rates are suppressed or
even reversed (Niinemets et al. 2011). In general, static systems should be avoided
and a dynamic flow-through chamber or enclosure used, such that the analyte
concentrations in both inflowing (Cin ) and outflowing (Cout ) air are measured. The
differences between these, together with information on flow rate (v) and leaf area
(AL ) or mass, are then used to calculate emission rates:

v .Cout  Cin /
ED : (15.3)
AL

In reality, the calculation is somewhat more complex due to changes in water

vapour concentration (Niinemets et al. 2011). Dynamic enclosure systems have the
additional advantage of ease of control and recording of leaf temperature and pho-
tosynthetically active photon flux, both of which may affect BVOC emission rates.
The preferred method for measuring leaf-level emission rates is therefore the
use of portable photosynthesis systems with built in carbon dioxide and water
vapour gas sensors with a compatible leaf cuvette or chamber with temperature and
light control. The LI-Cor LI-6400 series of instruments is almost ideal, following
modifications to reduce adsorption of volatiles on system component surfaces, but
other suitable designs (including those made by PP Systems, ADC or Walz GmbH),
are available. Plumbing modifications might be necessary to allow for sub-samples
of the inflowing and outflowing chamber air to be diverted to a suitable BVOC
detection system. This might be an on-line detection system such as a proton-
transfer reaction mass spectrometer, or a sample preconcentrator, prior to transfer
to an off-line detector such as a gas-chromatography system.
Some studies have estimated emission rates at the branch scale, by enclosing
branches (or parts of branches) with leaves in a dynamic chamber (e.g., made of
Teflon film; Owen and Hewitt 2000; Owen et al. 2001) with internal measurement
of light intensity, and air or leaf temperature. However, these do not readily lend
themselves to the control of light and temperature and have largely been superseded
by the controllable leaf chambers described above.
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 435

15.3.5 Measuring Canopy-Scale Emission Rates

With the invention of fast-response sonic anemometers and fast-response and

sensitive detectors for BVOCs, it is now possible to make tower-mounted,
micrometeorologically-based, measurements of canopy-scale BVOC emission
rates. This avoids the need to extrapolate from leaf- or branch-scale measurements
to the canopy scale. However, these methods are only suitable in simple terrain with
a large upwind fetch of 100–1,000 m over reasonably homogeneous canopies. They
may be especially suitable for agroforestry plantations (e.g., Misztal et al. 2011) but
not for small patches of woodland (e.g., small urban parks).
One of the technically simplest methods of measuring canopy-scale emission
rates is relaxed eddy accumulation (REA) (e.g., Gallagher et al. 2000; Greenberg
et al. 2003). In REA measurements, ambient air is sampled at an appropriate height
above a uniform canopy at a constant flow rate. The vertical wind vector is measured
at high frequency and the sample air stream switched such that it passes into separate
sampling reservoirs for the upward and downward flowing air. Analysis of the
contents of the reservoirs is then carried out off-line. The disadvantage of the method
is that it gives a single flux estimate integrated over, say, one hour, and is generally
very labour-intensive (Ciccioli et al. 2003).
More recently, eddy covariance (EC) and its variant, virtual disjunct eddy
covariance (vDEC) have been developed and used for BVOC flux estimates. In EC,
co-located high frequency measurements of the vertical wind velocity and BVOC
mixing ratio are combined to calculate fluxes. Currently, only a few instruments
are capable for EC of BVOCs. For single BVOCs, these instruments include the
“fast isoprene sensor” (Guenther and Hills 1998) and the proton-transfer reaction
mass spectrometer with quadrupole mass spectrometer (PTR MS) which can
sample individual masses at 2 Hz (Karl et al. 2001). However, for simultaneous
measurements of multiple BVOC fluxes, only the protontransfer reaction time-of-
flight mass spectrometer (PTR TOF MS) (Jordan et al. 2009; Ruuskanen et al. 2011)
has high enough temporal resolution for EC measurements (see e.g., Müller et al.
2010).
In vDEC, the requirement for very high frequency BVOC measurements is
mathematically relaxed and the standard PTR MS can be used. The detector is used
to scan over a small suite of predetermined masses, which generates a discontinuous
or “disjunct” set of fast response data. These are then combined with the vertical
wind velocities to calculate fluxes, normally averaged over one hour or half an hour.
The method of vDEC has now been used extensively to measure BVOC fluxes from
both natural and managed vegetation, including an oil palm (Elaeis guineensis)
agroforest in Malaysia (Misztal et al. 2011). The next stage of development in
landscape-scale flux measurements is likely to result from the application of vDEC
using instruments mounted on low-flying slow aircraft.
Directly measuring canopy-scale emission rates from urban habitats is very
difficult, except from the largest of urban parks because of the need to have a
large upwind fetch over a homogeneous canopy. However, Langford et al. (2010)
436 S.M. Owen et al.

Fig. 15.1 Temperature responsiveness of isoprene (closed circles) and isopentane (open circles)
concentrations at the Marylebone Road automatic monitoring station, London, UK. The data are
based on 5 years of hydrocarbon measurements (modified from Langford et al. 2010). To estimate
the percentage of temperature-dependent isoprene concentration, scatterplots of isoprene vs.
benzene (marker of vehicle emissions) were derived for different temperatures, and the intercepts
of these relationships were used for the background concentrations of isoprene not attributable
to direct emissions from vehicles. The temperature-dependent percentage was calculated as the
ratio of the intercept at given temperature to the total isoprene present. To assess the effect of
temperature on evaporation, analogous relationships were derived for non-biogenic compound
isopentane. Error bars indicate the uncertainty of intercept values for the temperature bands
5–0 ı C, n D 114; 0–5 ı C, n D 3,405; 5–10 ı C, n D 9,539; 10–15 ı C, n D 12,176; 15–20 ı C,
n D 9,340; 20–25 ı C, n D 3,171; 25–30 ı C, n D 673; 30–35 ı C, n D 73. The regression line is
given by y D 1.8 C 3.0exp(0.11x) (r2 D 0.97, P < 0.0001)

used vDEC to measure the fluxes of a range of volatiles, including some emitted
by vegetation, over the city of London. They correlated fluxes with traffic flow to
estimate the vehicle- and non-vehicle-related components of the observed fluxes.
They also applied a novel analysis, using the long-term measurements of benzene
concentrations, to determine the temperature-dependent biogenic component of the
isoprene flux, shown in Fig. 15.1. At an ambient temperature of 25 ı C they estimated
that biogenic emissions of isoprene account for about 50 % of the observed
concentration of the compound in the middle of London. However, this percentage
drops significantly as ambient temperature falls and non-biogenic sources become
relatively more important.
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 437

15.4 Quantifying Emissions of BVOCs from Urban Regions

and Agroforestry Plantations

15.4.1 Quantifying Urban Emissions

As indicated above, estimating the BVOC flux from urban vegetation poses a
number of challenges. In the urban environment, vegetation is fragmented and trees
from very many species are present, including both native and exotic species, as
individual specimens in domestic gardens or common areas, as linear features along
streets, canals and railways, as groups in squares, gardens, allotments, or other
smaller open spaces, or in larger numbers in parks, community woodlands or other
large non-built areas. In size, they vary from small shrubs and saplings in gardens or
new plantings, to fully grown specimens which may reach 40 m or more in height.
The number of different species present in an urban area varies with land-use type,
sub-urban residential areas typically containing many more exotic species than, for
example, dense inner-city areas.

15.4.1.1 A Case Study

A simple extrapolation, multiplying leaf area based emission factors by leaf area
index (LAI), and applying scaling factors depending on light and temperature
dependencies was used by Owen et al. (2003) to estimate BVOC flux from the West
Midlands, UK urban area. To estimate canopy emission potential per unit ground
area for a particular BVOC compound at standard conditions of temperature of
30 ı C and light of 1,000 mol m2 s1 , the following model was used:

i Dk
X
Fpot D Epsi Mi Sf;i (15.4)
i D1

where k is the number of plant species within the canopy, Fpot is the habitat emission
potential at standard conditions of temperature and light, Epsi is the BVOC emission
potential for species i (g g1 dry mass h1 ), Mi is plant species biomass per
ground area (g m2 of ground) and Sf,i is the fractional contribution of species i to
total canopy area. As discussed in Sect. 15.3.1, and Guenther (2013), this approach
provides a rough estimate of canopy emission as it neglects the within-canopy
variation in emission potentials and environmental drivers, and does not consider
shading of vegetation by neighbouring plants and buildings. Nevertheless, all this
information may be very difficult to obtain in urban environments due to numerous
unique radiative transfer situations (Osmond 2010).
As described in Sects. 15.3.2 and 15.3.3, simulation of urban emissions requires
estimation of the species fractional composition in the study area. This can be done
by remote sensing (Sect. 15.2.3) or ground survey method (Sect. 15.3.3), although
438 S.M. Owen et al.

combined approaches are also possible. Owen et al. (2003) used a ground survey
method (Sect. 15.3.3) for the 900 km2 West Midland (UK) area, which is described
in more detail by Owen et al. (2006) and Donovan et al. (2011).

15.4.1.2 Assigning Emission Factors to Species Contributing

to the Canopy

Once the dominant species composition for each urban land-class is known, BVOC
emission factors for each species can be extracted from databases or literature,
taking care to note the environmental conditions for which the emission factors
are defined, and the methods used to derive them. Where no literature exists, it
is possible to use values from members of the same genus or family (Benjamin
et al. 1996) although allocating emission factors in this way is likely to be prone
to errors and large uncertainties. This can be demonstrated by the Quercus genus
whose members either emit isoprene or monoterpenes, at different rates and with
different chemical speciation. It is therefore better to sample as many species as
possible to obtain missing emission factors.

15.4.1.3 Extrapolating to Canopy Flux Estimates

The relative contribution of each species to the urban land-class canopy, and the
LAI of each species can be used to weight the relative contributions of species
emissions to the urban land-class canopy to give canopy-averaged emission factors
which can then be used with climate data in emission flux algorithms and models
to predict average flux of each compound from the canopy. This approach does not
account for different emission factor values throughout the canopy nor for extinction
of light through the canopy, but it has also been used by Owen et al. (1997, 2001)
for the Mediterranean regions, and Pierce and Waldruff (1991) for continental US.
More advanced schemes with simplified canopy models (e.g., Sellers et al. 1992
big leaf approach) have been employed in Guenther et al. (1995, 2006, 2012) and
Lavoir et al. (2011) to model regional and global BVOC fluxes. Using the simplified
canopy model, Lavoir et al. (2011) obtained a simple relationship between canopy
monoterpene emission and a remotely-sensed spectral vegetation index for spatially
heterogeneous vegetation cover. MODIS satellite data were used to obtain the
fraction of light absorbed by the canopy at 1 km2 resolution. Nevertheless, simple
big-leaf models tend to overestimate the flux (Dai et al. 2004) and for correct
integration of fluxes, either layered models (Guenther et al. 2006, 2012; Niinemets
et al. 2013) or two-big-leaf models (de Pury and Farquhar 1997; Dai et al. 2004)
should be used.
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 439

15.4.1.4 Assessing the Biogenic Component to Total Urban Volatile

Emissions

Hundreds of hydrocarbon compounds are detected in the air over towns and cities.
Measurements of BVOC fluxes show mainly compounds of anthropogenic origin,
from traffic, industrial and residential emissions. Isoprene is commonly detected
in city air, but it is not necessarily emitted from the vegetation planted within
and around the urban area. It has been shown that exhaust from vehicles is a
source of isoprene (Borbon et al. 2001; Langford et al. 2010). Several methods
are used to distinguish between anthropogenic and biogenic isoprene in cities. A
primary indication of a biogenic source is an increase of concentrations/flux during
the summer months. A diurnal peak of isoprene concentrations during the rush
hours in winter indicates the anthropogenic contribution. If this is assumed to be
constant, then the biogenic contribution in summer can be estimated by subtracting
the winter concentrations (Borbon et al. 2001). A further quantification of biogenic
isoprene can be made by calculating the isoprene/acetylene ratio. Traffic emissions
are thought to be the major source, if not the only source, of acetylene in urban
air. A constant ratio over time indicates the same origin for both compounds.
Durana et al. (2006) adopt the same approach, using the ratio of isoprene/iso-butene
concentrations to estimate the biogenic contribution to isoprene.
Different studies report different biogenic contributions to urban isoprene con-
centrations. von Schneidemesser et al. (2010) report that biogenic isoprene is not
significant in London and Paris. Langford et al. (2010) separated the biogenic
fraction of isoprene in London air using 5 years of hydrocarbon data collected
between 2001 and 2006 by the Hydrocarbon Network monitoring station situated
on Marylebone Road using regressions of isoprene against benzene concentrations
(a marker of vehicle emissions) at different temperatures, and used the isoprene
intercept of the regression as an estimate of biogenic emissions (Fig. 15.1). Langford
et al. (2010) found only slight increases in iso-pentane concentrations relative to
benzene at higher temperatures due to increased evaporative emissions, but the
temperature-dependent fraction of isoprene was large and increased exponentially
with temperature. Throughout year, 19 % of isoprene at the London site was
estimated to originate from biogenic sources, but this percentage was much greater
on warmer days (Fig. 15.1).

15.4.2 Quantifying Emissions of BVOCs from Agroforestry

Plantations

Because agroforestry plantations generally are monospecific, it is more straight-

forward to quantify BVOC emissions from these canopies, compared with the
urban canopies described in Sect. 15.4.1. Plantation canopies lend themselves more
easily to direct canopy-scale flux measurements. For example, BVOC fluxes have
been measured directly from oil palm (Elaeis guineensis) (Misztal et al. 2010,
440 S.M. Owen et al.

2011) and from rubber tree (Hevea brasiliensis) plantations (Baker et al. 2005).
The UN Food and Agriculture Organisation (FAO) publishes annual information
on global crops and forestry plantations, including statistics and lists of globally
important agroforestry plantation species (e.g., FAO 2006). The plantation species in
Table 15.3 are extracted from FAO (2006), and represent the world’s most important
agroforestry plantation species. Three types of emissions and flux estimate methods
are presented in Table 15.3: (i) canopy-scale flux measurements, (ii) leaf- or
branch-scale species emission rate measurements, and (iii) leaf or branch-scale
emissions extrapolated to canopy flux estimates. Table 15.3 shows a wide range
of plantation species’ BVOC emission rates, and canopy-scale flux measurements
and extrapolated estimates. It is remarkable that many of the world’s important
agroforestry plantation species are high BVOC emitters. Elaeis guineensis, Salix
spp., Populus spp. and Eucalyptus spp., for example, are all strong BVOC emitters.
This has implications for regional air quality, and this is already being addressed
in recent research to provide low BVOC-emitting Populus (Behnke et al. 2012;
Rosenkranz and Schnitzler 2013). However, to our knowledge, there are no emission
measurements to date for important plantation species such as Cunninghamia spp.,
Tectona spp. and Ziziphus spp.

15.5 Uncertainties and Challenges for Future Research

15.5.1 Uncertainties

As we have previously mentioned, extrapolations carry high uncertainty when they

are derived from field survey data based on a stratified random sample using an
urban land-use classification to estimate foliar and whole tree biomass, leaf area
and leaf area index for individual urban land class kilometre squares. Further
uncertainties arise when these estimates of urban forest attributes are used to
characterize the typical BVOC emission and gaseous dry deposition potentials of
each land class kilometre square and thus entire urban regions. Errors are therefore
propagated, and should be estimated and presented with extrapolated results. A
method for doing this is described in Donovan et al. (2011), and is based on
simple first principals of uncertainty analysis. Donovan et al. report the following
uncertainties associated with extrapolating their field measurements and survey
data to urban regional scale in West Midlands conurbation of UK: leaf area per
tree ˙ 35 %; foliar biomass per tree ˙ 60 %; foliar biomass per km2 ˙ 70 %; foliar
biomass for the West Midlands ˙ 120 %; projected crown area per tree ˙ 25 %; tree
cover per km2 ˙ 40 %; tree cover in the West Midlands ˙ 95 %; West Midlands
BVOC emission estimate ˙ 240 %. Uncertainties associated with the top-down
approach include uncertainties with the model assumptions made, and instrumen-
tation uncertainties. Niinemets et al. (2011) give a comprehensive analysis of the
uncertainties associated with extrapolating from leaf and branch measurements to
15 Scaling Emissions from Agroforestry Plantations and Urban Habitats 441

canopy-scale fluxes. In fact, comparisons between canopy-scale isoprene emissions

based on leaf-level emission measurements extrapolated with a canopy environment
model and above-canopy flux measurements suggest that discrepancies are less
than the uncertainty associated with these two approaches (Guenther et al. 2006;
Niinemets et al. 2013).

15.5.2 Future Challenges

With increasing urbanization, rapid land-use change driven by expansion of food

and biofuel crop production, and changing climate, it will be increasingly important
to monitor, estimate and predict the resulting changes in BVOC emissions, as these
cause changes to atmospheric chemistry and climate. It is important that urban
greening or de-greening and planting of biofuel crops is done in an environmentally
responsible manner, which includes a consideration of the BVOC emission potential
both at the time of planting and in the future. The research challenges include
making better use of remote sensing data that are becoming available at higher
spatial and spectral resolutions. Methods for interpreting these data using inverse
modelling and by direct spectral analysis to estimate BVOC emissions are in their
infancy, but are improving. Future progress in BVOC emissions and effects research
depends on the development of more efficient methods for ground-truthing of
biophysical parameters, and on simple, fast chlorophyll fluorescence techniques
to indicate photosynthetic status and stress within individuals. Further progress
also critically depends on and more sensitive and rapid analytical methods for
determining BVOC concentrations and fluxes. Much has been achieved, but much
more work is required to understand these complex processes in an increasingly
urbanized world.

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Chapter 16
Global Modelling of Volatile Organic
Compound Emissions

Kirsti Ashworth, Christophe Boissard, Gerd Folberth, Juliette Lathière,

and Guy Schurgers

Abstract The majority of volatile organic compounds emitted from the terrestrial
biosphere (BVOCs) are highly reactive hydrocarbons that have been shown to affect
atmospheric composition across the full range of temporal scales from fractions of
seconds to centuries and spatial scales from m to global. Furthermore, biogenic
emissions are thought to account for around 90 % of the total quantity of non-
methane hydrocarbons released into the atmosphere each year. As a result, BVOCs
have substantial air quality and climate impacts, and there is an urgent need to
quantify and map their emissions as precisely as possible. In this chapter we
outline the use of computer models to estimate annual global emissions of BVOCs
and the on-going efforts to validate and constrain the output from such models.
The current generation of BVOC emission models generally includes only the
constitutive emissions of a handful of compounds: chiefly isoprene, monoterpenes
and methanol, which are thought to account for about 80 % of the total flux from
the biosphere. At present, it is estimated by global models that total annual emission
of isoprene amounts to around 500 Tg of carbon, with the emissions dominated
by tropical ecosystems and by tree species. The emissions of monoterpenes are

K. Ashworth
Institute for Meteorology and Climate Research – Atmospheric Environmental Research
(IMK-IFU), Karlsruhe Institute for Technology (KIT), Kreuzeckbahnstrasse 19, 82467
Garmisch-Partenkirchen, Germany
C. Boissard • J. Lathière ()
Laboratoire des Sciences du Climat et de l’Environnement – LSCE-IPSL, CEA-CNRS-UVSQ,
L’Orme des Merisiers, 91191 Gif-sur-Yvette, France
e-mail: [email protected]
G. Folberth
Met Office Hadley Centre, Exeter EX1 3PB, Devon, UK
G. Schurgers
Department of Physical Geography and Ecosystem Science, Lund University,
Sölvegatan 12, 223 62 Lund, Sweden

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 451
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 16,
© Springer ScienceCBusiness Media Dordrecht 2013
452 K. Ashworth et al.

similarly distributed, although high levels of monoterpene emissions are also seen
from the boreal forests. There is currently no consensus on the annual estimate
of monoterpene emission, with estimates ranging from 30 to 150 Tg of carbon.
Apart from these main compounds, the biosphere emits many hundreds of different
compounds, some of which are produced as a short-lived, transient response to stress
rather than as constitutive emissions. We discuss the role that biogenic emissions of
reactive trace gases play in the Earth system as a whole, and consider the potential
feedbacks that exist between BVOC emissions, atmospheric composition, air quality
and climate, and the terrestrial biosphere, and how these can be studied with Earth
system models. We finally suggest ways of improving and further developing the
global models.

Abbreviations

ANN Artificial Neural Networks

ATP Adenosine triphosphate
BER Basal emission rate
BVOCs Biogenic volatile organic compounds
CCMs Chemistry-climate models
CCN Cloud condensation nuclei
CTMs Chemistry-transport models
DMADP Dimethylallyl diphosphate
ESMs Earth system models
GCMs General circulation models
NADPH Nicotinamide adenine dinucleotide phosphate
PPFD Photosynthetic quantum flux density
PFTs Plant functional types
SOA Secondary organic aerosols
VOCs Volatile organic compounds

16.1 Introduction to Global Modelling

Biogenic volatile organic compounds (BVOCs) constitute a major sink for the OH
radical, the atmosphere’s most powerful oxidant, particularly over land. Emissions
of BVOCs, thus, mediate the oxidative capacity of the atmosphere, affecting the
atmospheric lifetime of other chemical species, such as methane. Biogenic volatiles
released into the atmosphere react rapidly with atmospheric oxidants: O3 , and the
OH and NO3 radicals (see e.g., Atkinson 2000; Atkinson and Arey 2003). Although
they may form peroxy radicals that go on to participate in ozone formation,
the products of these initiation reactions are often oxygenated species of much
16 Global Modelling of Volatile Organic Compound Emissions 453

lower volatility than the parent BVOC (Griffin et al. 1999). Such products can
partition into the particle or aerosol phase, either through direct nucleation or by
condensing onto existing particles (see e.g., Hallquist et al. 2009 and references
therein). Detailed analyses of the composition of aerosol particles, through the
use of carbon isotopes for example, have shown that the majority of their mass
is of biogenic origin, even in highly polluted regions (Zhang et al. 2007; Jimenez
et al. 2009). This is not captured with current atmospheric chemistry and aerosol
models, which are known to underpredict the concentration of biogenic secondary
organic aerosol (SOA) almost everywhere (Heald et al. 2005). Biogenic volatiles,
and particularly isoprene, also play a key role in the distribution of reactive nitrogen
in the atmosphere through the formation of organic nitrates (von Kuhlmann et al.
2004; Ito et al. 2009), especially peroxyacetyl nitrate (PAN). PAN is a long-
lived compound, with an atmospheric lifetime of a few months in the cold-free
troposphere.
Modelling studies suggest that including isoprene emissions in atmospheric
chemistry models increases the methane lifetime by over 20 % as compared to
model simulations with no isoprene (Pike and Young 2009). There is currently
considerable uncertainty over the role of isoprene as a sink for the OH radical in
very low NOX environments. Flux and concentration measurements taken during
field campaigns in the Amazon suggest that the OH radical is somehow “recycled”
during the chain of reactions involving isoprene (Lelieveld et al. 2008). Various
novel chemical mechanisms have been proposed to account for this (see e.g., Peeters
et al. 2009; Paulot et al. 2009), as has the concept of segregation, or lack of mixing,
of isoprene and the OH radical (Butler et al. 2008). As yet, none of the existing
mechanisms accounts fully for the observed ambient OH radical concentrations (see
e.g., Pugh et al. 2010). Although the uncertainty in the mechanism is confined to
low-NOX regions, the impact is felt globally, with Archibald et al. (2010) reporting
that inclusion of an “OH-recycling” mechanism in a global atmospheric chemistry
model resulted in a 14 % reduction of the projected methane lifetime.
Biogenic volatiles are therefore a crucial component and have to be considered
when investigating the evolution of the Earth system, especially in the context of
globally changing climate, land use and landcover, and atmospheric composition.
In this chapter, we first review the different modelling approaches used so far to
estimate emissions of BVOCs such as isoprene, monoterpenes or other emitted
species (Sect. 16.2), addressing particularly the question of model evaluation
(Sect. 16.3). The different feedbacks involving BVOCs in the context of the Earth
system study and modelling will be detailed in Sect. 16.4, with special attention to
feedbacks with methane, ozone, secondary organic aerosols or changes in diffuse
radiation, involved not only in the atmospheric composition but also in climate
forcing. Finally, perspectives and challenges to take global modelling forward
and find new approaches for BVOC emission estimates, and future experimental
investigation needs are presented in Sect. 16.5.
454 K. Ashworth et al.

16.2 Modelling the Emissions from the Terrestrial Biosphere

Ground-breaking work by Chameides et al. (1988) demonstrating the importance

of BVOCs on local- to regional-scale air quality resulted in increasing efforts
to quantify the emissions of these compounds. This work soon expanded in
scale from regional to global and in approach from detailed inventories based
on laboratory measured fluxes to parameterizations for use in computer-based
simulation models (Pierce and Waldruff 1991; Guenther et al. 1995, 2006). Initially,
these parameterizations for light and temperature responses were empirical fits to
laboratory leaf-level emissions (see e.g., Guenther et al. 1991), based on observed
photosynthesis-like response curves (Monson et al. 1992). More recently, biochem-
ical techniques such as isotope labelling studies have improved understanding of
the synthesis routes involved in the production of BVOCs within plants (see e.g.,
Fuentes et al. 2000), permitting a more process-based approaches to be adopted.
There are currently two main ways to modelling the emissions of BVOCs
from vegetation: models that were developed to empirically represent the observed
responses to environmental drivers – here referred to as empirical models – and
models that were developed based on theoretical understanding of the underlying
processes – here referred to as process-based models (Grote and Niinemets 2008).
Although these two types of models are fundamentally different, they can be param-
eterized to describe a similar degree of variance in large-scale models (Keenan et al.
2009; Niinemets et al. 2013). So, there are often no objective criteria for choosing
a model for present-day simulations (Guenther 2013; Niinemets et al. 2013). For
predicting emissions under global change when the predictions necessarily need
to be extrapolated beyond the existing data, process-based models might be more
useful (Arneth et al. 2007).
Here we focus on isoprene and monoterpenes as only limited data are available on
emission rates of other BVOCs. The fluxes of “other” BVOCs are usually estimated
using the same exponential temperature relationship as that for monoterpenes,
although the temperature dependence of the emissions of many BVOCs is not
known with any certainty (Guenther et al. 1995, 2012).

16.2.1 Empirical Isoprene Emission Models

Laboratory studies show that leaf-level isoprene emissions are strongly dependent
on leaf temperature and light levels. Guenther et al. (1991) demonstrated clearly
that the light dependence of the emissions closely matched the light dependence
of photosynthesis, which had been earlier linked to electron transport (Farquhar
et al. 1980). Thus the photosynthesis algorithms were modified to simulate isoprene
emissions.
These algorithms were further developed into an empirical model (Guenther
et al. 1995) that could be applied worldwide with refined coefficients, but still
16 Global Modelling of Volatile Organic Compound Emissions 455

based on measurements on a limited number of plant species (Guenther 2013).

Thus, it was acknowledged that in this early application, basal emission rates
(BERs) for five basic plant functional types (PFTs), and derived ecosystem-level
emission estimates, had a large uncertainty (Guenther et al. 1995; Guenther 2013).
Appropriate foliage densities were also assigned to these ecosystems. In addition,
a simple canopy model was incorporated to determine the fraction of sunlit and
shaded leaves within the canopy in order to calculate leaf-level photosynthetically
active radiation based on the radiation reaching the top of the canopy.
Currently, the two most widely used empirical models are MEGAN (Guenther
et al. 2006, 2012) and BEIS (Pierce and Waldruff 1991). They are briefly described
below. For further detail, we refer to the original publications and Guenther chapter
in this volume (Guenther 2013).
The Model of Emissions of Gases and Aerosols from Nature (MEGAN) is
an empirical emission model developed at the National Center for Atmospheric
Research (NCAR) that can be used to simulate the fluxes of 19 different groups
of BVOCs (Guenther et al. 2006, 2012). While the basic mathematical forms of the
temperature and light responses for isoprene developed by Guenther et al. (1995)
were retained, the emission algorithms were extensively modified in an attempt
to better capture seasonality through the inclusion of an activity factor dependent
on leaf age. In addition, studies indicated that plants acclimate to their growth
environment, responding both to instantaneous changes in light and temperature,
and to past light and temperature environment. This environmental response was
divided between an initial rapid phase and longer-term phase that lasted for a
period of ca. 10 days (Guenther et al. 2006), and the temperature and light response
algorithms were adapted to incorporate these rapid and longer-lasting effects.
The Biogenic Emissions Inventory System (BEIS) (Pierce and Waldruff 1991)
is a high-resolution regional-scale empirical emission model that uses the same
light and temperature dependence algorithms as those formulated by Guenther et al.
(1995). Developed by the scientists of the US Environmental Protection Agency
(EPA) to generate an emission inventory for the USA, it used highly detailed land
cover maps, and species- and location-specific values of base emission rates for 230
land-use types (Pierce et al. 1998). The canopy model incorporated in BEIS (Gay
1987) also differed from that in MEGAN (Guenther et al. 2006, 2012; Guenther
2013 for a comparison).
Despite the similarity in approach and the same algorithms employed by the
two models, the emission estimates from BEIS and MEGAN often vary widely
due to the differences in landcover, emission factors and canopy models (see e.g.,
Warneke et al. 2010; Carlton and Baker 2011; Hogrefe et al. 2011; Guenther
2013). In general, comparisons between the models, and between models and
observations suggest that MEGAN tends to overestimate and BEIS underestimate
BVOC emissions (Carlton and Baker 2011), with MEGAN isoprene emission
estimates more than a factor of two higher than BEIS (Warneke et al. 2010; Carlton
and Baker 2011; Hogrefe et al. 2011). Carlton and Baker (2011) also found MEGAN
methanol emissions to be more than four times higher than the estimates by BEIS.
To date, the models have not been found to outperform each other consistently when
456 K. Ashworth et al.

coupled to an air chemistry model and compared with measurements of a range

of atmospherically relevant chemical species (e.g., ozone, aerosols, formaldehyde)
and across a series of sites (see e.g., Warneke et al. 2010; Carlton and Baker 2011;
Hogrefe et al. 2011).

16.2.2 Use of Neural Networks for Biogenic

Isoprene Emissions

Artificial neural networks (ANNs) constitute a special type of empirical models.

They have been shown in numerous studies to describe complex sets of environ-
mental interactions. The existing emission algorithms mainly focus on more rapid
variations in isoprene emission and do not consider acclimation over more than
10 days (Guenther et al. 2006). Nevertheless, lower frequency variations, e.g.,
seasonal changes in tree capacity to release isoprene have been observed to be
responsible for a significant, in some cases a major, part of the overall emission
fluctuations, reaching up to three orders of magnitude (Monson et al. 1994; Geron
et al. 2000; Boissard et al. 2001; Petron et al. 2001). If not correctly assessed, this
low frequency variability can represent a major source of discrepancies in isoprene
emission assessments (Guenther et al. 1995). Furthermore, the impacts of some
environmental parameters, such as water availability, are still not taken into account
in these models, due to the complexity of the processes involved.
ANN is a statistical approach to calculate non-linear regressions between the
output data, here BVOC emission rates, and a set of relevant input data, environ-
mental parameters (Fig. 16.1). Among the other available statistical methods, ANNs
present the advantage of being the most parsimonious, and as with other non-linear
regression methods, ANN is not overly sensitive to co-linearity among different
explanatory variables (Bishop 1995; Dreyfus et al. 2002). In simulating BVOC
emissions, ANNs were first employed by Lasseron (2001), Simon et al. (2005a) and
Boissard et al. (2008). Boissard et al. (2008) assessed variations in isoprene emission
rates using environmental parameters integrated over a few days to a few weeks prior
to the measurements using the emission datasets specifically built for the study. A
total of 9 high (instantaneous) to low (up to 3 weeks) frequency regressors were
obtained, which together accounted for up to 91 % of the variability in isoprene
emission rate (compared to 42 % when the G95 algorithm was employed for the
same dataset). The obtained isoprene emission algorithm was mainly sensitive to
air temperature cumulated over 3 weeks (T21) and to instantaneous light intensity
L0 and temperature T0 variations. T21, T0 and L0 alone accounted for 76 % of the
overall variability.
Using a similar approach Simon et al. (2005a, b) coupled isoprene and monoter-
pene emissions measured from Amazonian tree species with physiological and
environmental regressors. ANNs have also been used to provide km-scale emission
maps of European forest carbon fluxes (Papale and Valentini 2003), and to improve
assessments of biogenic soil NOX emission variations (Delon et al. 2007).
16 Global Modelling of Volatile Organic Compound Emissions 457

Fig. 16.1 Schematic representation of the structure and functioning principles of an artificial
neural network (ANN) for isoprene emission rate purposes using a multi-layer perceptron (MLP)
method (a feedforward artificial neural network model). Within an ANN, a different number of
neurons Nj can be used and arranged in a network of different layers. Whatever the network
(here based on two neurons), every input regressor xi (for instance environmental parameters)
is connected to each neuron Nj , all are connected together and the final output value, Ycalc , is
jP
  
DN P
iDn
calculated according to Ycalc D w0 C wj f w0;j C wi xi , where w0 is the initial
j D1 iD1
connecting weight between the bias and the output, N the number of neurons Nj , f the transfer
function (a parameterized bipolar, non-linear function), w0,j the initial connecting weight between
the bias and the neuron Nj , wi the connecting weight between the input and the neuron Nj , and xi the
input regressor. Optimised weights are assessed during a training phase, which consists, starting
from random values of wj , in minimising the difference E between the calculated and expected
outputs (for instance measured isoprene emission rates). In this figure, E is calculated as follows:
P 
kDz 2
ED Ycalc  Yexp where k is the number of the z output values
kD1

16.2.3 Process-Based Isoprene Emission Models

Process-based models for leaf-level isoprene emission are based on the biochemical
processes underlying the synthesis of isoprene from the carbon assimilated during
photosynthesis. As described in previous chapters (Li and Sharkey 2013; Monson
2013), isoprene is produced in plant chloroplasts where energy from sunlight is
converted to chemical energy in the form of ATP and NADPH for use in carbon
fixation and reduction. There are two different pathways for isoprenoid synthesis
that both depend critically on the amount of energy available from ATP and NADPH
458 K. Ashworth et al.

(Niinemets et al. 1999). The bulk of volatile isoprenoids released from plants
are synthesized via the plastidic 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-
xylulose 5-phosphate pathway (Lichtenthaler 1999). Thus, the synthesis of these
isoprenoids is strongly linked to chloroplastic electron transport rate (Rasulov et al.
2009), undergoing a series of catalytic conversions, via key intermediate compounds
such as glyceraldehyde 3-phosphate and dimethylallyl diphosphate (DMADP), to
produce the wide variety of hydrocarbons, including isoprene and other terpenes.
The four process-based isoprene emission models currently in use assume that
isoprene production is intrinsically and quantitatively linked to a single or several
rate-limiting steps in precursor-forming reactions, and furthermore, that isoprene is
released immediately, i.e., isoprene emission rate is equal to the rate of isoprene
production. An overview of these models is provided by Arneth et al. (2007).
Due to the complexities of the biochemical pathways involved and the high
degree of species-specificity of some of the processes and reactions, only one of the
available process-based algorithms has been incorporated in global-scale emission
models (Arneth et al. 2007; Pacifico et al. 2011). The Niinemets et al. (1999) model
assumes that the rate-limiting step is the production of DMADP from ATP and
NAPDH, which are in turn limited by the rate of electron transport within the
chloroplast. The Niinemets et al. (1999) model assumes that the use of electron
flow for isoprene production competes with the electron transport required for the
assimilation of carbon during photosynthesis, and the model calculates the rate of
synthesis, and therefore emission, of isoprene, assuming that a certain fraction of
photosynthetic electron transport is diverted to the production of isoprene. The
hypothesis has been recently revised in that it is not the competition between
photosynthesis and isoprene emission per se, but low effective Michaelis-Menten
constant for isoprenoid synthesis pathway leading to strong control of emissions by
electron transport rate under physiological conditions (Rasulov et al. 2009, 2011).
The emission flux of isoprene, I, is given by (Niinemets et al. 1999; Arneth et al.
2007):

I D ©J˛ (16.1)

where J is the photosynthetic electron transport rate, ’ is the electron cost for
isoprene production, and © is an empirically derived fraction of photosynthetic
electron transport that is used for isoprene production. As the parameter © represents
the activity of the isoprene synthesis pathway in the chloroplasts, it is strongly
dependent on temperature and the chloroplastic concentration of carbon dioxide. In
this model, initially developed for strong isoprene emitters Liquidambar styraciflua
and Quercus spp. (Niinemets et al. 1999), the electron fraction also depends on
isoprene synthase activity, and thus, can vary with species as well as with leaf long-
term environment (Monson et al. 2012; Grote et al. 2013 for more details).
As our understanding of the biochemical processes and reactions involved in
the synthesis of isoprene and its intermediates improves, so does the prospect of a
fully process-based model capable of reproducing the production and emission of
isoprene and higher terpenoids without the need for species-specific parameters.
16 Global Modelling of Volatile Organic Compound Emissions 459

16.2.4 Monoterpene Emission Models

In most monoterpene emitting vegetation, monoterpenes are stored in specialized

storage structures referred to as pools. These take a variety of forms, including
leaf cavities (Eucalyptus), glandular cells (Lamiaceae), resin canals (Pinus) and
ducts (Abies) in conifer needles (see e.g., Fuentes et al. 2000; Loreto and Schnitzler
2010). As a result, emissions of monoterpenes in species with storage structures are
decoupled from their synthesis; the emission rate depends on the rate at which the
monoterpene volatilizes from the pools and diffuses through the stomata (Fuentes
et al. 2000; Grote and Niinemets 2008; Schurgers et al. 2009). Hence, monoterpene
emissions are generally dependent only on temperature according to a simple
exponential relationship (Guenther et al. 1995, 2012) in the form:

E D Es e “.T Ts / (16.2)

where Es is a species-specific basal emission rate (BER), Ts is the temperature at

standard conditions, typically taken 30 ı C, and “ is a scaling exponent linking the
observed monoterpene emission rate to temperature.
Apart from evaporation from storage, several tree species emit monoterpenes
entirely (e.g., Quercus ilex, Staudt and Seufert 1995; Fagus sylvatica, Dindorf
et al. 2006) or partly (e.g., Pinus sylvestris, Taipale et al. 2011) light-dependently
similarly to isoprene. To capture this, the empirical isoprene emission algorithm
(Sect. 16.2.1) or the process-based isoprene model (Niinemets et al. 2002, 2013)
have been employed to simulate monoterpene emissions. For global-scale studies,
the MEGAN model version 2.1 (Guenther et al. 2012) applies varying fractions of
light-dependent emissions for the different monoterpenes.
A more explicit approach to account for storage of monoterpenes within the plant
has been developed by Schurgers et al. (2009), based on a detailed model by Bäck
et al. (2005) that explicitly describes monoterpene synthesis and diffusion from the
site of synthesis to ambient atmosphere (Niinemets and Reichstein 2002, 2003). The
authors (Schurgers et al. 2009) assume that monoterpene synthesis and emission are
decoupled. A fraction of the synthesized monoterpenes is emitted directly, and the
remainder is stored in specific structures referred to as storage pools. Emissions
from these storage pools are regulated by the size of the pool and a temperature-
dependent residence time, such that:

Eemis D m=£ (16.3)

where Eemis is the monoterpene emission rate from the storage pool, m is the size
of the storage pool (g m2 ) and £ is the average residence time (days), which
also has a temperature dependence. The introduction of the storage pool into
the emission algorithms improved the day-to-day pattern of emissions, leading to
reduced variability as the pool acted as a buffer between production and release; the
model also better described longer-term variations in the emissions as seasonality
was much better represented (Schurgers et al. 2009).
460 K. Ashworth et al.

16.2.5 Estimates of Global Fluxes

The first estimate of global biogenic isoprene flux was 250 Tg C year1 , calculated
by Müller (1992) for the IPCC 3rd assessment report. Guenther et al. (1995) re-
visited this, systematically assigning emission factors to global ecosystems and
combining these with high resolution (0.5ı by 0.5ı ) global vegetation distribution
and meteorological data, and arrived at a total of 503 Tg C year1 , but with a
very large degree of uncertainty of a factor of three. The standard configuration
of the MEGAN model (Guenther et al. 2006, 2012) estimates an average annual
isoprene emission of 600 Tg isoprene year1 , or 530 Tg C year1 for different
model parameterizations. Varying driving variables such as meteorology, vegetation
distribution and leaf area index datasets yielded estimates ranging from 440 to
660 Tg C year1 (Guenther et al. 2006).
In contrast, a review by Arneth et al. (2008a) on isoprene emission estimates for
“present-day” (1990s) conditions generated by the Guenther et al. (1995) algorithms
showed an apparent convergence to 520 Tg C year1 , with a range of only
460–600 Tg C year1 , in spite of a wide range of driving variables. Analogously,
Guenther et al. (2006) found that total emissions varied between 11 and C29 %
(from a “standard” run of 500 Tg C year1 ) when the MEGAN algorithms were
driven with different meteorology, vegetation distributions or leaf area index data.
However, Arneth et al. (2008a) argue that given the uncertainty in the basic
driving variables, e.g., vegetation distribution and incoming radiation, as well as
the assumption of the global validity of emission factors extrapolated from very few
enclosure and field campaign measurements, such consensus is not warranted. In
particular, comparisons of simulated emissions against measured fluxes (see e.g.,
Müller et al. 2008; Hewitt et al. 2009; Warneke et al. 2010) unanimously show
large discrepancies in both the magnitude and temporal fluctuations (especially
longer-term seasonal variations). Arneth et al. (2008a) suggest that there is an
urgent requirement for the global biogenic emission modelling community to
systematically tackle the sources of uncertainties, such as our understanding of the
process of synthesis and emission of isoprene, as well as addressing unknowns,
such as emission factors for some biomes. Even a full mechanistic understanding of
factors controlling emissions at the leaf and stand level, however, will not eliminate
all the uncertainties from calculations of global emissions.
Arneth et al. (2011) reported the findings of an emission model intercomparison
study involving three of the most widely used and extensively evaluated global
vegetation and emission models: MEGAN (Guenther et al. 2006), LPJ-GUESS
(Arneth et al. 2007) and BVOCEM (Lathière et al. 2010). In their “standard” set-
ups, average annual emissions for the period 1981–2002 were 378 Tg C year1 ,
463 Tg C year1 and 496 Tg C year1 respectively, slightly lower than those
generated for the 1990s or 2000s from the respective models due to the lower global
average temperature of the 1980s. However, when model inputs were exchanged
(e.g., LPJ-GUESS driven with NCEP meteorology as used with MEGAN, etc.),
not only did their estimates for total global emissions diverge, but the spatial
16 Global Modelling of Volatile Organic Compound Emissions 461

pattern of distributions was altered in ways that cannot be fully reconciled with
the community understanding of emissions (Arneth et al. 2011). Furthermore,
Ashworth et al. (2010) demonstrated that global emissions could vary by up to
32 % (and local emissions by up to 77 %) simply due to altering the time-resolution
of the input meteorology. The introduction of a representation of the circadian
control of base emission rate observed over a Borneo oil palm plantation (Hewitt
et al. 2011) reduced total global isoprene emissions by as much as 21 % (but see
Keenan and Niinemets 2012). These studies collectively not only highlight our
incomplete understanding of the isoprene emission process, but also suggests that
our confidence in a value of 500 Tg C year1 may be somewhat premature.
Differently from isoprene, there is no consensus about the total global flux of
monoterpenes, with estimates ranging from 30 to 150 Tg C year1 (Arneth
et al. 2008a; Guenther et al. 2012). It is unclear why monoterpene emission
estimates should reflect the uncertainties in driving variables more closely than
similar estimates for isoprene, although it has been suggested that greater range in
monoterpene simulations reflects outlying observations using problematic parame-
terizations (Guenther 2013). Thus, convergence of isoprene models is likely to be a
result of less variation in the parameter sets used in modelling isoprene fluxes.
The empirical and process-based methods for isoprene and monoterpene emis-
sions explained above can be regarded as two alternatives for the same research
questions, and they can be parameterized to result in similar fluxes. However,
MEGAN and LPJ-GUESS have differences not only in the emission algorithm, but
also in the way vegetation coverage is described. MEGAN, the “empirical” model,
comes with a more detailed vegetation description than used in LPJ-GUESS, the
“process-based” model, where the description of vegetation is limited to a relatively
small number of plant functional types. However, LPJ-GUESS allows for dynamic
changes in the vegetation distribution, and is therefore more representative than
more detailed but static vegetation distributions in addressing climate change effects
on BVOCs.

16.3 Model Evaluations

Measuring and analysing volatile organic compounds emitted by the terrestrial

biosphere is essential to improve our understanding of the nature and quantity
of chemical species emitted, together with the variability and sensitivity of their
emissions to environmental parameters. All global simulation models share the use
of basal emission rates (or emission capacities) as the basic way to parameterize
vegetation emissions. Although the algorithms in use have developed additional
parameterizations to consider effects resulting, for example, from meteorological
history (Guenther et al. 2006), seasonal development (Arneth et al. 2008b) or
leaf age (Guenther et al. 2006), a common and major drawback of all methods
is the missing representation of the observed variability between different stands,
individual trees or even branches (Niinemets et al. 2010a; Bäck et al. 2012). The
462 K. Ashworth et al.

basal emission rates, as key parameters of the models, also bear large uncertainties,
which directly affect the simulated emissions. In order to overcome this problem,
large-scale (ecosystem-scale and upward) measurements of emission capacities
would be needed. However, determination of the emission capacities at higher scale
faces practical problems. Measurements in a controlled environment, as can be done
with leaves or branches, are practically impossible, and within-canopy interactions,
e.g., between vegetation, micrometeorology, and within-canopy chemistry, would
need to be represented properly. Here the ways of conducting direct ecosystem and
biome level emissions measurements and proxies for deriving emission estimates
are analysed.

16.3.1 Field Measurements

Field campaigns conducted around the world such as BEMA (Seufert et al. 1997),
BOREAS (Pattey et al. 1999), BIPHOREP (Laurila and Lindfors 1999), PROPHET
(Westberg et al. 2001), ECHO (Spirig et al. 2005), ESCOMPTE (Simon et al.
2005a, b) or OP3 (Hewitt et al. 2010) provide fundamental qualitative and quan-
titative information improving our capability of testing, evaluating and constraining
the BVOC emission models. Flux measurements, performed by different techniques
such as gradient profile, relaxed eddy accumulation, disjunct eddy covariance and
eddy covariance (Guenther et al. 1996; Ciccioli et al. 2003; Müller et al. 2010),
are of particular importance for the direct evaluation of BVOC emission models.
Field measurements therefore provide a unique opportunity to test, under different
environmental and climatic conditions, the representativeness of BVOC emissions
simulated by models for different ecosystems.
In order to evaluate the performance of global models on timescales of seasons
to decades, long-term measurements that are run for several years are particularly
needed, and currently scarce. Global models capture variability at timescales of
hours to weeks well, but knowledge on both the short, episodic events (e.g.,
emissions related to insects or wind damage) and the long-term changes (seasonal
developments and adaptation to changes in climate and CO2 concentration) are
currently poorly addressed in ecosystem-scale measurements and consequently
poorly represented in global models.

16.3.2 Use of Satellite Data to Constrain the Emission Models

In parallel with the development of these so-called “bottom-up” methods of

estimating and evaluating emissions of BVOCs, efforts have been made to use a
“top-down” approach to constrain model estimates. The top-down approach is an
indirect method that makes use of satellite measurements of relevant atmospheric
constituents to back-calculate the flux of the parent BVOCs. It is not possible
16 Global Modelling of Volatile Organic Compound Emissions 463

to measure reactive BVOCs such as isoprene directly by satellite as atmospheric

concentrations are too low and the spectral lines are broad and overlapping with
other atmospheric constituents (see e.g., Palmer et al. 2003), so longer-lived short-
chained reaction products that can accumulate at higher concentrations and have
sharper spectral lines are used as a proxy. This work has not only led to constraints
on total biogenic emissions, but has also highlighted sources of uncertainties in
bottom-up flux estimates, advancing understanding of the operating processes (see
e.g., Barkley et al. 2009).
Formaldehyde (HCHO) columns have been extensively used for the purpose
of constraining isoprene emissions from satellite observations (see e.g., Palmer
et al. 2003 , 2006; Barkley et al. 2008, 2009; Shim et al. 2005) and identifying
seasonal and spatial variations (Abbot et al. 2003; Barkley et al. 2009; Harrison
et al. 2013). HCHO is a useful proxy for isoprene as it is formed rapidly from both
isoprene and its direct oxidation products, and has a sufficiently short atmospheric
lifetime itself to ensure that it is not transported far from the original source of
isoprene (Palmer et al. 2003). In addition, relatively little HCHO is formed from
other BVOCs. Monoterpene oxidation products tend to partition to the aerosol
phase, and while methanol does oxidize to form HCHO, it does so over a much
longer timescale and forms part of the background signal, in theory allowing it
to be distinguished from isoprene-derived HCHO (Shim et al. 2005). Satellite
derived measurements of formaldehyde columns are, however, themselves subject
to significant uncertainties on the order of 100 % (e.g., Palmer et al. 2006), and
require an atmospheric chemistry model inversion or back-trajectory to deduce
the required isoprene source strength. Thus, uncertainties in the knowledge and
understanding of isoprene oxidation chemistry (see e.g., Prather et al. 2001; Butler
et al. 2008; Lelieveld et al. 2008; Archibald et al. 2010) affect the ability of the
“top-down” approach to accurately map HCHO to isoprene (Palmer et al. 2006).
Direct comparisons between “top-down” estimates of isoprene emission and
those generated by the MEGAN algorithms (Guenther et al. 2006) show good
agreement in North America (Abbot et al. 2003; Palmer et al. 2006), and South
and East Asia, with the exception of China (Fu et al. 2007). These comparisons also
suggest that MEGAN significantly overestimates isoprene emission in the tropics
(Fu et al. 2007; Barkley et al. 2008). HCHO columns appear to show better seasonal
agreement with direct measurements of both isoprene and HCHO over both North
and South America (Palmer et al. 2003; Barkley et al. 2008) than do the MEGAN
estimates, although it should be borne in mind that HCHO columns are subject to
a great deal more spatial and temporal smearing than the “bottom-up” estimates
by models. Shim et al. (2005) estimated global isoprene emission on the basis of
HCHO columns, arriving at a value of 566 Tg C year1 , i.e., an excellent agreement
with estimates derived from the emission algorithms.
The use of glyoxal retrievals is being developed to further constrain emissions
of isoprene and other BVOCs, as well as their atmospheric reactions (see e.g.,
Stavrakou et al. 2009). Work is on-going to elucidate links between other satellite
retrievals, e.g., aerosol optical depth (Veefkind et al. 2011), CO columns (Jiang et al.
2011), and emissions of other BVOCs such as monoterpenes.
464 K. Ashworth et al.

Apart from HCHO, other longer-lived volatiles such as methanol can also
be measured by remote sensing. Stavrakou et al. (2011) used satellite derived
observations of total methanol column to estimate the source strength of methanol
from the terrestrial biosphere. Their calculation of 100 Tg year1 was in close
agreement to the estimate of 105 Tg year1 generated by the MEGAN algorithm,
although analysis of the spatial and temporal discrepancies between the two led to
the development of improved MEGAN algorithm for methanol emission (Stavrakou
et al. 2011).

16.4 Use of Global Models to Analyse the Feedbacks

in the Earth System

16.4.1 Overview of Feedbacks with BVOC

A number of impacts and feedbacks among BVOC emission, vegetation activity

and climate potentially occur (Fig. 16.2), and some of these feedbacks are currently
being included in state-of-the-art Earth system models (ESMs, Kulmala et al.
2013 for detailed analyses of the impact of global feedbacks). These feedbacks
link the terrestrial biosphere with the chemical composition of the atmosphere
and, consequently, with climate. Central to these feedbacks are the production,
emission and photochemical oxidation of BVOCs. The key processes that influence
or are influenced by the production of BVOCs in plants, their emission to the
atmosphere and their subsequent photochemical oxidation include the formation
and removal of ozone, nitrogen compounds and secondary organic aerosols as well
as the methane lifetime and burden in the troposphere. BVOC emissions are also
impacted by drought and wildfires affecting the distribution and composition of
vegetation, assimilation of CO2 and evapotranspiration. Since BVOCs can directly
and indirectly influence climate via various forcing processes (e.g., methane, ozone,
SOA) these processes form multiple feedback cycles between the biosphere and the
atmosphere. Arneth et al. (2010) have given a detailed review of the processes and
feedbacks that are outlined in Fig. 16.2. Many of the processes outlined in Fig. 16.2
have been recognized only very recently and are still associated with very large
uncertainties.
BVOCs emitted to the atmosphere interact with anthropogenic emissions to im-
pact atmospheric composition. Formation of ozone and SOA directly affect climate
by altering the atmospheric radiation budget (Myhre et al. 2013). BVOCs also
compete with methane for OH radicals and thus can increase methane atmospheric
lifetime, the methane burden in the troposphere and, ultimately, the climate because
methane is the third most important atmospheric greenhouse gas (Voulgarakis et al.
2013). The resulting changes in surface temperature directly affect BVOC emissions
closing the feedback cycle (Kulmala et al. 2013).
16 Global Modelling of Volatile Organic Compound Emissions 465

Fig. 16.2 Biogeochemical feedbacks linking biosphere and atmosphere via emission of biogenic
volatile organic compounds (BVOC). Ozone and particulate matter (PM) – especially secondary
organic aerosols (SOA) – that are produced through atmospheric oxidation influence plant
productivity (diffuse radiation, ozone damage to plants). BVOC emissions also significantly
influence atmospheric composition and climate, for instance, the lifetime of greenhouse gases
such as methane (£CH4 ). On a broader scale, climate and air composition have an impact on
the biosphere via deposition of chemical species (e.g., nitrogen species) and growing conditions
such a temperature, solar radiation intensity and water availability. Extreme events such as heat
waves and droughts promote wildfires which in turn impact on plant productivity and atmospheric
composition by emission of ozone precursor species and aerosols. Changes in plant productivity
due to biosphere-atmosphere feedbacks also affect exchange processes between the biosphere and
the atmosphere such as evapotranspiration and assimilation of CO2 , ozone and other atmospheric
species that either promote or hamper plant productivity. From this picture it becomes clear that
biogeochemical feedbacks represent a strong link between the biosphere and the atmosphere with
important implications for atmospheric composition and climate

A second and potentially equally significant pathway in this feedback cycle is

via the interaction of ozone with plant productivity and BVOC formation. Ozone
is a potent oxidant and is harmful to plant tissue. Ozone enters the plants via the
stomata, and plants react to elevated ozone concentrations by reducing stomatal
conductance (Sitch et al. 2007). This has major consequences for CO2 and water
vapour exchange, ultimately affecting carbon assimilation and evapotranspiration,
but also for most of the trace gases (Calfapietra et al. 2013 for detailed discussion;
Harley 2013).
Such feedback cycles centered around the production and emission of BVOCs
affect a number of other processes which could result in multiple feedbacks.
Photochemistry of BVOCs, for instance, leads to the formation of SOA that has
the potential to change the radiation flux in the atmosphere. Secondary organic
aerosols influence the actinic flux and scatter incoming radiation, increasing the
diffuse fraction of short-wave radiation, and this is expected to increase plant
466 K. Ashworth et al.

productivity (Mercado et al. 2009). SOA also participate in formation of cloud

condensation nuclei (CCN) affecting cloud droplet number density, cloud lifetime
and precipitation (Carslaw et al. 2010).
Earth system models (ESM) are used to understand these feedbacks and their
implications for climate and air quality on both regional and global scales. Many
processes are still beyond the grasp of ESMs due to limited understanding.
Furthermore, even for processes that are currently included, estimates of their
importance in terms of impact and feedback strength vary largely with systematic
studies only just beginning (see e.g., Lee et al. 2011). Here, we will make no attempt
to present further details on the uncertainties connected with feedbacks involving
BVOCs, but will focus instead on recent studies that have looked at some of the
individual processes.

16.4.2 Impacts of BVOCs on Methane

Methane is an important greenhouse gas, atmospheric concentration of which has

increased from 380 ppbv (Monnin et al. 2001) at the last glacial maximum to
715 ppbv in 1750 (Etheridge et al. 1998) and 1,787 ppbv in 2008 (Dlugokencky
et al. 2009). The global mean atmospheric abundance of CH4 is determined by the
interplay between emissions and sinks. CH4 emissions are very diverse, covering
a wide range of natural (wetlands, termites, oceans, marine hydrates, geological
sources, wild animals, and wildfires) and anthropogenic (energy, mining, landfills
and waste treatment, ruminants, rice agriculture, and biomass burning) sources
(Denman et al. 2007).

16.4.2.1 Competition Among Methane and BVOC for OH Radicals

Oxidation of CH4 by OH radicals represents the main atmospheric sink for methane.
However, BVOCs of both natural and anthropogenic origin other than methane
(also called non-methane volatile organic compounds, NMVOCs) compete for the
available OH radicals, thereby altering the oxidizing capacity of the atmosphere
(Levy 1971; Hauglustaine et al. 1998; Bey et al. 2001; Collins et al. 2002; Folberth
et al. 2006). Through this interaction, NMOVCs have a significant impact on the
chemical lifetime of CH4 in the atmosphere. Globally, BVOCs dominate by far the
total amount of NMVOCs emitted into the atmosphere (Guenther et al. 1995)
Generally, it is expected that volatile compounds deplete OH in an unpolluted
environment, thereby increasing the chemical lifetime of CH4 (Granier et al. 2000;
Lelieveld et al. 2002; von Kuhlmann et al. 2004). This perturbation to the CH4
lifetime will be amplified by the CH4 feedback on its own lifetime (Prather et al.
2001). However, in a polluted environment, where NMVOC emissions are generally
collocated with pronounced emissions of NOX , NMVOC oxidation leads to a
16 Global Modelling of Volatile Organic Compound Emissions 467

buildup of ozone and, consequently, OH formation by the catalytic action of NOX .

This widely accepted picture has recently been challenged by Lelieveld et al. (2008),
who suggested that oxidation of natural NMVOCs, notably isoprene, might provide
a mechanism for OH recycling even in a pristine, low-NOX environment. This
proposed mechanism could effectively limit the impact of primary NMVOCs on the
CH4 lifetime. Thus, it could have significant implications for the future evolution of
the CH4 atmospheric burden, but the mechanism of OH recycling remains largely
unclear.

16.4.2.2 Climate Change and Methane-BVOC Interactions

Recent modelling studies have shown that BVOC emissions could increase substan-
tially due to climate change (Sanderson et al. 2003; Lathière et al. 2005). Lathière
et al. (2005) calculated an increase in total annual BVOC emissions by 75 % from
725 Tg C year1 at present-day conditions to 1,250 Tg C year1 at the end of the
twenty-first century (Fig. 16.3). An increase in BVOC emissions of this magnitude
has the potential to significantly affect the methane lifetime, and consequently the
methane burden. O’Connor et al. (2010) gave a very rough estimate of the impact
of a 75 % increase in BVOC emissions on the methane lifetime and compared this
impact to the change in methane lifetime due to climate warming. They came to
the conclusion that an increase in BVOC emissions of this order and the chemical
kinetic effect on the methane lifetime are of similar magnitude, but opposite in sign
and could potentially cancel each other out.
These studies did not take into account the effect of increased atmospheric
concentration of CO2 on the emissions of BVOC. Monson et al. (2007) showed
that higher than present-day levels of atmospheric CO2 can significantly reduce or
even completely abolish the effect of climate warming on emissions of isoprene.
Modelling studies by Arneth et al. (2007) and Heald et al. (2009) have found little or
no impact on future emissions of isoprene in relation to climate warming due to the
CO2 inhibition effect. Pacifico et al. (2012), including the effect of CO2 inhibition
on isoprene emission, calculated the change in methane lifetimes due to changes in
isoprene emissions between the present and the end of the twenty-first century. They
found a very small decrease in the methane lifetime of only 2 %. However, other
studies demonstrate that depending on growth conditions, elevated CO2 actually
might enhance the emissions, especially if this is coupled to enhanced leaf area
index (Sharkey et al. 1991; Sun et al. 2012), suggesting that any global modelling
of elevated CO2 effects on isoprene emission is bound to large uncertainty.
It is currently not fully clear whether the emissions of other BVOCs such
as, for instance, terpenes are also sensitive to elevated levels of atmospheric
CO2 concentration. Increasing, decreasing or constant emissions in response to
elevated CO2 have been observed, and the effects apparently differ for different
monoterpenes (Loreto et al. 2001; Staudt et al. 2001).
468 K. Ashworth et al.

Fig. 16.3 Distribution of annual emissions (g C m2 month1 ) of isoprene (upper panels) and
monoterpenes (lower panels) for present-day (left) and future (right) scenarios according to
Lathière et al. (2005). BVOC emissions are calculated using the ORCHIDEE global vegetation
model including parameterizations from Guenther et al. (1995). The present-day simulation uses
1990s climate forcing from CRU (Climate Research Unit, UK). The future simulation employes
the climate reconstructed by the LMDz general circulation model for 2100s for an atmospheric
CO2 concentration of 560 mol mol1 . The inhibition effect of increasing atmospheric CO2
concentration on isoprene emissions is not taken into account in this work (Wilkinson et al. 2009;
Sun et al. 2012)

16.4.2.3 Methane-BVOC Interactions: Lessons from the Past

In addition to model simulations, we may learn about the future evolution of the
interactions between BVOC emissions and atmospheric methane concentrations
by turning to the past. Studying past atmospheric CH4 fluctuations from ice cores
allows for some of the vast uncertainties around the magnitude of the CH4 -NMVOC
feedback in future climate to be constrained. Atmospheric CH4 concentrations have
increased by more than 65 % from the last glacial maximum (LGM), 21,000 years
before present, and to the preindustrial era (Chappellaz et al. 1993, 1997; Brook
et al. 2000; Valdes et al. 2005; Kaplan et al. 2006; Harder et al. 2007). This has
been attributed to a substantial increase in BVOC emissions from a more productive
tropical forests and the developing boreal forests as a consequence of the shrinking
of the continental ice sheets (Valdes et al. 2005; Kaplan et al. 2006; Harder et al.
2007).
16 Global Modelling of Volatile Organic Compound Emissions 469

Valdes et al. (2005) and Kaplan et al. (2006) have calculated an increase of the
CH4 lifetime from the last glacial maximum to the preindustrial holocene due to the
significant increase in BVOC emissions over the same time period. They found an
increase in the methane lifetime by approximately 1.3 years (19 %) and 2.1 years
(29 %) from 7.1 (Valdes et al. 2005) and 7.3 (Kaplan et al. 2006) years at last
glacial maximum to 8.4 (Valdes et al. 2005) and 9.4 (Kaplan et al. 2006) years,
respectively, at the preindustrial Holocene (PIH) as a consequence of increasing
BVOC emissions. The estimates of the two groups are based on different chemistry-
climate models and BVOC emission models. In those two studies, BVOC emissions
were calculated to increase by 100 % (Valdes et al. 2005) and 57 % (Kaplan
et al. 2006) over the same time period. This increase in CH4 atmospheric lifetime
would account for between 55 and 88 % of the increase in the atmospheric CH4
concentration from LGM to PIH (Valdes et al. 2005; Kaplan et al. 2006). However,
these model studies have large uncertainties since many effects cannot be taken into
account at the current stage of model development. Nevertheless, these modelling
studies have found some experimental support recently (Loulergue et al. 2008).
Because of its inherently non-linear nature, it is not possible to extrapolate the
past feedback directly into a future atmosphere. Nevertheless, it seems legitimate
to assume that a similar feedback process as in the past will result in a feedback of
the same sign and possibly similar magnitude in the future.

16.4.3 Impacts and Feedbacks of BVOCs, SOA

and Diffuse Radiation

16.4.3.1 Role of BVOCs in SOA Formation and in Cloudiness

Photochemical oxidation of BVOCs, in particular isoprene and terpenes, leads to

semivolatile organic compounds that can then partition into the particulate phase
to form SOA. Observations indicate that in many places around the world, more
than half of the submicron aerosol mass is actually organic (Zhang et al. 2007). The
organic material in these aerosols seems to be dominated by compounds of biogenic
origin (Hallquist et al. 2009). For example, particle growth rates in boreal forests
correlate with seasonal variation in primary productivity (Kulmala et al. 2004).
However, the global budget of secondary organic aerosols remains very uncertain.
Current best estimates of 12–70 Tg year1 (Kanakidou et al. 2005) may even be too
small by up to an order of magnitude (Hallquist et al. 2009). A limited understanding
of the relative contribution of biogenic SOA precursors and their atmospheric
photo-oxidation mechanisms as well as the magnitude of their emissions from
the terrestrial biosphere represents the dominant contributor to the uncertainties
around formation of secondary organic aerosols. Other important factors include
condensation, heterogeneous chemistry and evaporation of semivolatile oxidation
470 K. Ashworth et al.

products of BVOCs on aerosol surfaces and even polymerization of these chemical

species (Fuzzi et al. 2006; Hallquist et al. 2009).
Emissions of SOA precursors such as isoprene and many terpene species are very
sensitive to temperature but also to levels of solar radiation, soil moisture, foliar
biomass and to varying degrees to several other environmental factors (Guenther
et al. 1995, 2006). Many of these environmental factors are likely to change in
response to global alterations in climate. Furthermore, the abundance of secondary
organic aerosols and their properties can also depend on the composition of the
atmosphere. Atmospheric composition, in turn, is sensitive to changes in climate
but also to the amount of anthropogenic volatile species and BVOCs. For example,
key environmental factors that determine the concentration of the OH radicals in the
atmosphere are temperature, water vapour content and solar irradiance. The latter
itself is affected significantly by the amount, structure and geographic distribution
of clouds on the global scale.
Oxidized organic aerosols dominate the submicron aerosol mass over a wide
range of continental environments (Kanakidou et al. 2005; Zhang et al. 2007).
Consequently, these organic aerosols, in fact, of both primary and secondary
origin, have a significant direct impact on the atmospheric radiation budget by
absorbing and backscattering of solar radiation. Furthermore, SOA have been found
to represent an important factor in the growth of particles up to the size where they
can act as cloud condensation nuclei (CCN) at tens of nanometres (Allan et al.
2006; Laaksonen et al. 2008). Via this route, SOA significantly contribute to indirect
aerosol effects on climate. Particle formation via conversion of gas-phase species
into the aerosol phase contributes between 5 and 50 % to the global mean CCN
concentration in the boundary layer (Spracklen et al. 2008). Recently, it has been
shown that emission of BVOCs may control particle growth (Bonn et al. 2009) but
they can also suppress particle formation (Kiendler-Scharr et al. 2009).
Models of aerosol microphysics and chemistry have become fairly complex in
recent years (e.g., Tsigaridis and Kanakidou 2003; Vignati et al. 2004; Mann et al.
2010; Bellouin et al. 2011). These models have been integrated into chemistry-
climate models (CCMs) and ESMs to investigate the impacts of SOA and resulting
feedbacks. In particular, aerosol models have been coupled interactively to BVOC
emission models and atmospheric chemistry models (e.g., Tsigaridis et al. 2006;
Bauer et al. 2010; Kulmala et al. 2013).

16.4.3.2 Influences of BVOCs on Diffuse Radiation Fraction

and Implications for Productivity

Another mechanism that has the potential to impact the production of BVOCs by
the terrestrial vegetation is the effect of changes in the fraction of diffuse radiation
on plant photosynthesis. Changes in cloud cover or atmospheric aerosol, the latter
arising from natural sources such as volcanoes or anthropogenic sources such as
fossil fuel use and biomass burning, can alter both the total photosynthetic quantum
flux density (PPFD) and its diffuse fraction. Aerosols emitted from wildfires or
16 Global Modelling of Volatile Organic Compound Emissions 471

secondary organic aerosols (SOA) produced in situ from oxidation of BVOCs can
also increase the diffuse PPFD fraction.
Changes in diffuse PPFD fraction can have major effects on plant photosynthesis.
In general, plant photosynthesis increases non-linearly with incident photosynthetic
quantum flux density (PPFD). A saturation point is often reached at light levels on
bright days during the growing season. Under clear-sky conditions only a fraction of
the canopy is directly exposed to sunlight while the rest of the canopy remains in the
shade. Under cloudy or hazy conditions, the incoming sunlight is subject to a higher
degree of scattering, and so the PPFD is more uniformly distributed. In fact, both
theoretical and observational studies have demonstrated recently that photosynthesis
can be more efficient under diffuse light conditions (e.g., Gu et al. 2003; Niyogi
et al. 2004; Oliveira et al. 2007). In a modelling study, Mercado et al. (2009) have
investigated the impact of changes in diffuse PPFD on the global land carbon sink
and estimated that “global dimming” between 1960s and 1999 has increased the
carbon sink by about 25 %.
The effect of changes of diffuse PPFD on BVOC production and emission
through increased photosynthetic activity is likely to lead to another globally active
feedback cycle between plant productivity, BVOC emission fluxes, atmospheric
photochemistry and (secondary) organic aerosols (Kulmala et al. 2013). Studies
coupling aerosol models interactively to biogeochemical cycles, cloud formation
and radiation fluxes have started to increase our understanding of how natural
aerosols respond to changes in climate and atmospheric composition (Kulmala et al.
2013). However, the Earth system models that are required for these studies are
still in their early phase. In addition, much of the immediate connections among
different processes are still so poorly understood that process-level description in
models is not yet possible. Nevertheless, models including these feedbacks at the
level of best understanding provide encouraging evidence of the major significance
of the feedback loops between global change, BVOCs and SOA (Kulmala et al.
2013).

16.4.4 Impacts and Feedbacks of Ozone Leaf Damage

Further environmental impacts on BVOC emissions currently under investigation

are ozone leaf damage due to exposure to surface ozone and reduced isoprene
emission as a consequence of severe drought (Pegoraro et al. 2004). Ozone affects
adversely both human health and vegetation with the adverse effects on plants first
identified in the 1950s. Ozone causes cellular damage inside the foliage, thereby
reducing plant photosynthetic rate, accelerating leaf senescence and requiring
greater resource allocation to detoxification and repair (Ashmore 2005). At present,
many regions worldwide already experience near-surface ozone concentrations that
are persistently higher than 40 ppbv. At these levels, ozone may already cause visible
damage such as leaf reddening and necrosis, and reduction in crop yields (Ashmore
472 K. Ashworth et al.

2005). A further increase in the tropospheric ozone concentration is expected over

the twenty-first century (e.g., Hauglustaine et al. 2005).
Tropospheric ozone enters plants primarily through the stomata. Its potential
effect on the emission of BVOCs is through its impact indirectly on plant primary
productivity and directly on stomatal openness which is expected to temporally alter
water-soluble BVOC emissions (Niinemets and Reichstein 2003; Harley 2013).
However, an increase in the production of BVOCs as a protection mechanism
against ozone damage has also been suggested (e.g., Sharkey et al. 2008; Vickers
et al. 2009). In the NOX -depleted leaf interior, BVOCs react with ozone leading to
depletion in ozone concentration thereby reducing the damage.
The uptake of ozone by plants via the stomata is in itself an important
ozone removal process (e.g., Folberth et al. 2006; Fowler et al. 2009). Hence,
the emission of BVOCs by plants, the subsequent formation of ozone through
photochemical oxidation of BVOCs initiated by OH or even ozone itself, the
removal of ozone through stomatal exchange fluxes and the subsequent damage
to leaf cells by ozone altogether form a closed feedback cycle that can influence
both ozone concentrations, at least close to the surface, and BVOC emission. In
addition, the ozone-BVOC feedback cycle has the potential to affect other important
atmospheric constituents, in particular CO2 and water vapour that are subject to
stomatal regulation. Thus, the ozone-BVOC feedback cycle could potentially have a
significant impact on climate by affecting CO2 assimilation by terrestrial vegetation
(Sitch et al. 2007) and even evapotranspiration.
Currently, the mechanisms that contribute to, and the strength of the ozone-
BVOC feedback cycle are poorly understood. Only one model study has incor-
porated the effect of ozone on plant productivity to assess the impact of ozone
on the carbon cycle (Sitch et al. 2007). To date, the link between ozone and
BVOC emissions is not well understood, although it potentially can have a major
significance at local and global scales (Lerdau 2007).

16.5 Perspectives and Challenges

While the global- and regional-scale emission models discussed above have the
capability of calculating emission rates of a wide range of BVOCs, most simulations
are confined to estimates of so-called constitutive emissions of the trace gases
believed to account for the majority of the reactive carbon flux from vegetation.
Although it has been suggested that many thousands of different BVOCs are
released from the biosphere (Goldstein and Galbally 2007), a recent highly detailed
inventory of BVOC emissions (Guenther et al. 2012) demonstrated that to the best
of our understanding, and in line with observations, a mere 11 compounds account
for over 80 % of the flux to the atmosphere each year. Four compounds are routinely
included in global emission estimates. These are isoprene, a generic monoterpene
(typically ’-pinene), methanol and acetone, accounting for nearly 75 % of the
estimated total flux (Guenther et al. 2012).
16 Global Modelling of Volatile Organic Compound Emissions 473

In order to have confidence that our models are producing realistic estimates for
BVOC emissions under present-day conditions that are accurate across a range of
temporal and spatial scales, and for the right reasons, requires not only a thorough
understanding of the physiological and phenological drivers of the synthesis and
emissions of these compounds but also an appreciation of the level of detail
necessary to achieve a suitable precision in model output. As a community, global
emission and atmospheric chemistry modelers are constantly confronted by the
question of how much detail is sufficient detail. While this is obviously governed
to a certain extent by the scientific question being addressed by the model, and its
spatial and temporal extent, we do not have the capability at present to include the
emissions of all compounds under all conditions. Nor should we strive to. However,
we must be certain that we have sufficient understanding of the compounds emitted
and their subsequent impacts on atmospheric composition, air quality and climate
to ensure that correct decisions can be made regarding the level of detail required to
answer the research question being addressed.
An estimate of the total annual global emission of a handful of BVOCs may be
sufficient for a global study focusing on long-term climate impacts on terrestrial
emissions. On the other hand, model simulations intended to provide air quality
forecasts for a limited area require a much more spatially and temporally explicit
representation of both emissions and atmospheric reactions. If we are to accurately
capture rapid fluctuations in atmospheric concentrations of critical pollutants, a
similarly precise level of detail is required from biogenic emission models. So, what
do we currently miss in our global models, and what do we need?

16.5.1 Facing the Uncertainties

The main challenge facing global modelling of BVOC emissions is to reduce

uncertainties and produce emission estimates that are robust enough to lend con-
fidence to calculations of future projections and hindcasts. Uncertainties in global
modelling arise from three main sources: internal variability (stability, resilience
to random perturbations and errors), model uncertainty and, for all but present-
day simulations, scenario uncertainties (Hawkins and Sutton 2009). Ultimately, the
greatest confidence in modelled emission estimates must be associated with a model
that is grounded on the fundamental processes governing the synthesis and emission
of these compounds, rather than on empirical fits to data. As discussed earlier in this
chapter, the best-developed and most widely used BVOC emission model represents
the latter, although it is based on the temperature and light responses similar to
photosynthesis (Guenther et al. 1995, 2006, 2012), while the process-based models
still require some empirical fitting to data (Grote et al. 2013). While both approaches
are able to a similar degree capture present-day emissions (Niinemets et al. 2013),
their robustness under past and future climate trajectories is uncertain.
In the case of BVOC emissions, internal variability may simply reflect our lack of
fundamental understanding of the processes governing the synthesis and emission of
474 K. Ashworth et al.

these compounds (Monson 2013). As discussed in the previous chapters (Fineschi

et al. 2013; Li and Sharkey 2013; Monson 2013), even the base emission rates for
isoprene, the most studied among the biogenic trace gases, and for monoterpenes,
the second most widely studies class of compounds, appear to fluctuate, even
between neighboring trees of the same species, for reasons that are currently
beyond our ability to explain (see e.g., Niinemets et al. 2010b; Bäck et al. 2012).
Comparisons between modelled and measured isoprene fluxes appear to show
emission factors that change due to time of day (Funk et al. 2003; Hewitt et al.
2011), season (Müller et al. 2008; Grote et al. 2010), geographical location and
position within the canopy (Niinemets et al. 2010b, 2011). These changes can be
partly explained by our current knowledge of the drivers of isoprene synthesis, but
full mechanistic understanding has not yet been reached.
Model uncertainties are a result of simplifications in the representations of BVOC
emission mechanisms, as well as other simplifications within the model itself. Each
of the process-based models that has been developed for simulating isoprene and
monoterpene emissions is based on a single process within the isoprene synthesis
pathway, e.g., electron transport (see Arneth et al. 2007 for an overview), rather than
integrating all the processes known to occur (Grote et al. 2013). In addition to this
simplification of the overall process description, the genuine unknowns, such as the
fraction of electrons used for isoprene synthase, are further parameterized through
empirical fits to data (Arneth et al. 2007). Furthermore, in order to apply these
models, developed from observations at the leaf level, to the ecosystem and then to
the regional and global scales, a limited number of plant functional types is used, in
spite of the known variation between synthesis and emission rates between species
(see e.g., Schurgers et al. 2011; Guenther et al. 2006). At the regional and global
scales, these uncertainties are further compounded by uncertainties in the input data,
such as global vegetation distribution, leaf area, and meteorology (Guenther et al.
2006; Arneth et al. 2011).
Quantification of these uncertainties is difficult, but as Sect. 16.2.5 demonstrates,
the effects can be very large in some cases, albeit the isoprene models tend to
converge to a “right” value. This remarkable agreement in total global emissions
within and between models does not extend to comparisons between modelled and
measured fluxes. For example, Müller et al. (2008) found that modelled hourly
fluxes at a site in the Northern mid-latitudes were, on average, 35 % lower than
the measured fluxes, while the emissions estimated in the Amazon were between
a factor of two and five times too high in the wet season. Langford et al. (2010)
reported emissions estimated with the MEGAN algorithms (Guenther et al. 2006)
over South-East Asian rainforest that were four times higher than observed fluxes,
but a factor of two lower than the peak midday flux of isoprene from a nearby
oil palm plantation (Misztal et al. 2011). Hewitt et al. (2010) showed that, in spite
of large discrepancies in emission rates and landcover data, two estimates of total
isoprene emission for the whole Borneo agree to within 5 %. However, there were
significant differences in the spatial distribution of the emissions (Hewitt et al.
2010), suggesting that global emission estimates may converge to similar values
due to compensation of errors.
16 Global Modelling of Volatile Organic Compound Emissions 475

Obviously, the predictions of future NMVOC emissions from the biosphere are
surrounded by extremely large uncertainties related to both the magnitude and sign
of the emission change in the future but also to ecosystem adaptation and distribu-
tion in the changing climate. Current knowledge only gives a wide range of possible
scenarios of BVOC emissions under future climate conditions spanning from no
change to a large increase at the end of the twenty-first century. These predictions
also depend on the climate scenarios used to extrapolate future climate conditions.

16.5.2 The Crucial Need for Observations

The large number of studies carried out over the last few years, including field
campaigns, laboratory experiments, satellite data integration and model develop-
ment, have significantly improved our knowledge regarding BVOCs. However, there
are still large gaps in data regarding the diversity, emission rate and variability,
and chemical reactivity of BVOCs emitted by the terrestrial biosphere, underlining
the crucial need for observations. The validation of regional and global models
developed for BVOC emission is essential, but also highly complex, because the
emissions are characterized by a strong spatial and temporal variability and a
high sensitivity to environmental conditions (including climate, ecosystem, and
atmospheric concentrations of trace gases such as CO2 ). Observations, both field-
and laboratory-based, under a range of conditions, are therefore highly valuable to
elucidate the major emission drivers and improve BVOC emission schemes, with
flux measurements being the only way to evaluate emission model performance
directly. However, the sparse data we do have are, in many cases, corrected to
“standard conditions” using the very same emission models we are trying to use for
evaluation of the measurements, thereby introducing further bias and uncertainty
into the process (Niinemets et al. 2010a, b for a discussion).
To perform a consistent evaluation, a full set of data acquired over long time peri-
ods and in a large variety of ecosystems and meteorological conditions, throughout
the tropics, and mid- and high-latitudes, are of special interest. Such data would
facilitate testing the model capacity to represent the spatial and temporal variabilities
in BVOC emissions. On top of the emission flux data, complementary information
and observations regarding vegetation characteristics (ecosystems considered, leaf
area, canopy height) and meteorology are essential to constrain the model with
the field site specifics. These evaluations are typically performed by comparison
of simulated emissions with observations from a number of different field sites.
Even when showing that model results lie in the range of field data, it should be
acknowledged that such model evaluation often leaves the modeler with the feeling
of unfinished and unsatisfying work. To be fully robust, the evaluation of large-scale
regional or global emission models should include the use of long-term, preferably
permanent, quality controlled, fully documented flux measurements, conducted with
standardized evaluation tools, for example, in line with the procedures developed for
476 K. Ashworth et al.

data from the FLUXNET network (a network integrating worldwide CO2 , water and
energy flux measurements, http://fluxnet.ornl.gov/).
There is also a need to bridge the gap in both measurements and models between
the instantaneous leaf-level processes and fluxes, and the canopy- or ecosystem-
scale fluxes. This gap is, in reality, bridged by a plant canopy, a complex structure
with a high level of heterogeneity of temperature, light and chemical composition
(e.g., Guenther 2013; Noe et al. 2012). Such heterogeneity drives rapid dynamic
chemical processes that are at best highly simplified in models. To what extent
should processes acting on short timescales be included? Again, this is likely to
depend on the location, time, and precise nature of the scientific question being
addressed, and requires systematic sensitivity studies and assessments coupled to
high-quality flux data.
Data for BVOCs or their oxidation product concentrations from field studies or
from satellite products offer an indirect but complementary approach to evaluate
BVOC emissions and atmospheric chemistry and transport models. Satellite data,
in particular, enable the evaluation of model performance on large spatial and
temporal scales. The main issue in the use of concentrations, rather than fluxes,
is that concentrations provide a proxy for integrated fluxes rather than instantaneous
fluxes, and reflect the balance between compound production and destruction. Thus,
use of concentrations requires inclusion of atmospheric chemistry schemes, thus,
adding a layer of uncertainty and complexity.
We conclude that in order to gain an understanding of the fundamental processes
behind the synthesis and emissions of BVOCs, and to address the question of how
much detail is indeed enough detail, we require more observations across larger
spatial and longer timescales.

16.5.3 Taking Global Modelling Forward

As outlined above, global models currently confine BVOC emission estimates

to so-called constitutive emissions, i.e., those compounds that are continuously
emitted under plant’s normal growing conditions. In addition to these compounds,
vegetation is known to produce a wide range of induced BVOCs, i.e., compounds
that are synthesized and emitted in response to both biotic and abiotic stresses, and
during particular phenological periods, for example during bud-burst or flowering.
Emissions under such conditions differ from constitutive emissions in both quan-
tity of carbon emitted and precise compound mix (Loreto and Schnitzler 2010;
Niinemets 2010). In some cases, a large number of different trace gases are produced
in small quantities. In others, a few compounds are released in very large quantities
(Loreto and Schnitzler 2010). In all cases, the importance of such emissions depends
on the precise compound mix, the size of the flux to the atmosphere, the timing of the
event (both in terms of time of day and time of year), the location of the flux and the
reactivity of the compounds involved (Arneth and Niinemets 2010; Niinemets et al.
2010a, b). While regular, well-documented events such as the emissions that occur
16 Global Modelling of Volatile Organic Compound Emissions 477

in response to mechanical wounding during forest or meadow harvesting periods

could be included in a model, sporadic, irregular events, such as herbivory would
be significantly more difficult to incorporate, although ways on incorporating such
emissions have been proposed (Arneth and Niinemets 2010).
Furthermore, can we be certain that the responses of vegetation to environmental
stresses observed now will continue in the future, or are the plants likely to adapt
to future changes? It has been observed that vegetation growing in hotter regions,
for example, displays a different temperature response than vegetation that naturally
grows in cooler areas, with a higher temperature optimum for BVOC emissions (see
e.g., Geron et al. 2006). It could perhaps be expected that, as temperature rises in the
future, vegetation will acclimate to this, with a slow shift in temperature response.
Similar changes could occur in responses to increasing atmospheric concentrations
of carbon dioxide or to changing patterns of precipitation or radiation, and clearly
again emphasize the need for more experimental data to bring modelling global
processes forward.

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Chapter 17
Climate Feedbacks Linking the Increasing
Atmospheric CO2 Concentration, BVOC
Emissions, Aerosols and Clouds in Forest
Ecosystems

Markku Kulmala, Tuomo Nieminen, Robert Chellapermal, Risto Makkonen,

Jaana Bäck, and Veli-Matti Kerminen

Abstract Biogenic volatile organic compounds (BVOCs) play a central role in

atmospheric chemistry via their high reactivity in the gas phase and via their
participation in atmospheric new particle formation and secondary organic aerosol
formation. The emissions of BVOC to the atmosphere depend on several climate-
related variables, making these compounds part of complex, yet potentially very
important, climate feedback mechanisms. Here we illustrated the role of BVOCs
in enhancing gross primary production (GPP) and cloud droplet number concentra-
tions. The first of these phenomena forms a positive feedback loop for the terrestrial
carbon sink (GPP feedback), whereas the second one forms a negative feedback
loop for the ambient temperature increase (temperature feedback).

17.1 Introduction

The atmosphere forms a major part of the environment that strongly impacts life
on the Earth. The atmosphere closely interacts with the biosphere, hydrosphere,
cryosphere and lithosphere on timescales from seconds to millennia. Changes in any
of these components are directly or indirectly communicated to others via intricately
linked processes, feedbacks, and interactions.
Recently, the importance of atmospheric aerosols to global radiation budget,
cloud formation, and alleged human health effects has motivated several inves-
tigations. Reactive gases, greenhouse gases and atmospheric aerosols are tightly

M. Kulmala () • T. Nieminen • R. Chellapermal • R. Makkonen • V.-M. Kerminen

Department of Physics, University of Helsinki, P.O. Box 64, 00014 Helsinki, Finland
e-mail: [email protected]
J. Bäck
Department of Physics, University of Helsinki, P.O. Box 64, 00014 Helsinki, Finland
Department of Forest Sciences, University of Helsinki, P.O. Box 27, 00014 Helsinki, Finland

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 489
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 17,
© Springer ScienceCBusiness Media Dordrecht 2013
490 M. Kulmala et al.

connected with each other not only via physical, chemical and meteorological, but
also via biological processes occurring in the atmosphere and at the atmosphere-
biosphere interface (Arneth et al. 2010; Carslaw et al. 2010; Mahowald 2011; Quinn
and Bates 2011). Human actions, such as emission policy, forest management and
land-use change, as well as various natural feedback mechanisms involving the
biosphere and atmosphere, have substantial impacts on the complicated couplings
between atmospheric aerosols, trace gases, greenhouse gases, air quality and climate
(Arneth et al. 2009; Raes et al. 2010; Shindell et al. 2012).
Anthropogenic emissions of greenhouse gases have increased substantially dur-
ing the past century. Elevated concentrations of CO2 and methane have been pointed
out as the most important forcing agents on climate during recent decades (IPCC
2007). However, it is not straightforward to describe the climate change in sufficient
detail, since there are several feedback mechanisms that are hard to understand
quantitatively. It has been recognized for decades that the biosphere plays an impor-
tant role in climate. It has also been suggested that the biosphere tends to regulate
and stabilise climate in order to keep it optimal for living organisms, as described
by the Gaia hypothesis (Lovelock 1979). Charlson et al. (1987) presented the so-
called CLAW-hypothesis, supporting the Gaia hypothesis. The CLAW hypothesis
connects ocean biochemistry and climate via a negative feedback loop involving the
ambient temperature, plankton activity, natural sulphur emissions from the ocean to
the atmosphere, cloud condensation nuclei production and cloud albedo change.

17.1.1 Setting the Scene: The Terrestrial CLAW Hypothesis

In terrestrial ecosystems, a number of potential climate feedback mechanisms have

been identified (Arneth et al. 2010) (see also chapter by Ashworth et al. 2013).
Here we focus on the continental CLAW hypothesis related to forest-atmosphere-
climate interactions proposed by Kulmala et al. (2004). We will investigate two
feedback loops associated with the continental CLAW summarized in Fig. 17.1.
Both these loops are initiated by increased CO2 concentrations, but are then
separated by two partly interacting branches. The central points of these two
loops are (i) the plant gross primary production (GPP) driven by photosynthesis
connected with the atmospheric CO2 concentration, and (ii) the ambient temperature
(T) which, on average, is higher in the world with larger CO2 concentrations.
Increased GPP and T are hypothesized to cause higher emissions of biogenic
volatile organic compounds (BVOC) which, as a result of atmospheric chemistry,
lead to increased secondary organic aerosol (SOA) concentrations. An increased
SOA concentration is expected to increase (i) the ratio between diffuse and global
solar radiation via enhanced scattering by aerosol particles, and (ii) cloud droplet
number concentrations (CDNC) via increased concentrations of cloud condensation
nuclei (CCN). Increased diffuse radiation fraction closes the GPP-loop via a positive
feedback for the carbon sink, while increased CDNC in turn closes the T-loop via a
negative feedback for the ambient temperature increase. While plausible, neither of
these two loops has been quantified, nor firmly proved, so far.
17 Climate Feedbacks Linking the Increasing Atmospheric CO2 . . . 491

Fig. 17.1 Two feedback loops associated with the continental CLAW hypothesis driven by
increased atmospheric CO2 concentration. The loop above is the clear-sky loop and the one below
is the cloudy-sky loop. Here T is the ambient temperature, GPP is the gross primary production,
BVOC refers to the biogenic volatile organic compounds, SOA to the secondary organic aerosol,
CS is the condensation sink (Eq. 17.1), Atot is the total aerosol surface area and Vtot is the total
aerosol volume, CCN refers to the cloud condensation nuclei, and CDNC is the cloud droplet
number concentration

17.1.2 Components of the Terrestrial CLAW Hypothesis

The plant gross primary production (GPP) is the difference between the net
ecosystem exchange of CO2 (NEE) and the total ecosystem respiration (TER).
In the boreal forest zone, photosynthesis occurs predominantly in sunlight during
the growing season (Hari and Mäkelä 2003) and is inhibited in winter. Forest
ecosystems are usually sinks of CO2 , and a direct negative feedback (the higher
the CO2 concentration, the higher the rate of photosynthesis, reducing CO2 in the
air) exists between increasing atmospheric CO2 concentrations and photosynthesis.
On the other hand, a positive feedback exists between ecosystem respiration
and temperature. At higher temperatures, water becomes a more important factor
influencing both GPP and TER.
Terrestrial vegetation contributes substantially to emissions of BVOCs, and these
emissions are closely related to photosynthesis (Fuentes et al. 2000). The ratio of
BVOC emission to carbon assimilation is generally a few percent (Guenther et al.
1995; Grace and Rayment 2000). These emissions serve many functions in plants,
and may vary between plant species (Niinemets et al. 2010). BVOCs are emitted as
mixtures of a variety of compounds depending on the seasonal and diurnal metabolic
activity of the plants. The most important compounds with respect to atmospheric
chemistry are terpenoids, i.e., isoprene, monoterpenes and sesquiterpenes. These
compounds can be synthesized in both aerial and belowground plant parts, and
large storage pools of some compounds are found in e.g., conifer foliage, trunks and
roots. The turnover rates of the terpenoid pools depend on both prevailing synthesis
level and factors controlling their evaporation. The biosynthesis of terpenoids
is regulated either by the supply of substrates, by the availability of energy, or by
492 M. Kulmala et al.

enzyme activities in the metabolic branching points or end-points of the biosynthetic

pathways (Bohlmann et al. 1998; Fischbach et al. 2002; Dudareva et al. 2004)
(Li and Sharkey 2013; Rajabi Memari et al. 2013; Monson 2013). Diffusion of
compounds from the site of their synthesis and storage to plant surfaces and
further to atmosphere is proportional to the concentration differences between the
components of the pathway and conductance along the diffusion pathway. Emission
of many BVOCs (including many non-oxygenated mono- and sesquiterpenes) is
not controlled by stomatal opening, and therefore they evaporate from the surfaces
at a compound-specific rate, as a function of temperature (e.g., Copolovici and
Niinemets 2005; Grote and Niinemets 2008; Grote et al. 2013; Harley 2013).
In the short term, both isoprene and monoterpene emissions show a clear temper-
ature dependence owing to the exponential relationship between their volatility and
temperature. Isoprene emissions are connected to irradiance as well because of the
close link between the isoprene emission and electrons derived from photosynthetic
light capture (Niinemets et al. 1999). Furthermore, the observed de novo synthesis
of monoterpenes indicates that prevailing light conditions can also be important
for their biosynthesis even in terpene-storing species such as several conifers
including pine (Pinus spp.) and spruce (Picea spp.) species (Shao et al. 2001;
Ghirardo et al. 2010), but especially in non-storing species such as Mediterranean
evergreen oaks (e.g., Quercus ilex, Q. coccifera, and Q. suber) (Staudt and Seufert
1995; Loreto et al. 1996) and some temperate broadleaf trees such as European
beech (Fagus sylvatica) (Dindorf et al. 2006). In addition to the short-term drivers,
emissions are responsive to medium-term changes in growth conditions, which
may affect the emission capacity, the shape of the light response as well as the
temperature optimum of emission (Sharkey and Loreto 1993; Lerdau and Throop
2000; Wilkinson et al. 2009; Heald et al. 2009; Grote et al. 2013; Monson 2013).
Effects of climate change on terpenoid emissions, and especially the effect
of elevated atmospheric CO2 concentration, have obtained increasing attention in
recent years. Abundant literature on the direct effect of elevated CO2 concentration
shows that, in general, isoprene emissions tend to decline with the rise of CO2
concentration (e.g., Wilkinson et al. 2009; Possell and Hewitt 2011, but see
also Sun et al. 2012). In contrast, studies of mono- and sesquiterpene emission
responses under elevated CO2 are rather scarce. Based on the available evidence,
it is likely that monoterpene emissions may not be as responsive to changes in
CO2 concentration as isoprene emission (e.g., Constable et al. 1999; Loreto et al.
2001). Also, increases in monoterpene emissions due to a simultaneous exposure
to elevated CO2 and temperature have been reported (Staudt et al. 2001; Räisänen
et al. 2008).
In the longer term, emissions of isoprene and monoterpenes may increase
significantly when the projected changes in plant species composition, vegetation
productivity and leaf area index (LAI) or density (LAD) associated with the climate
change are taken into account (Heald et al. 2009). The CO2 -driven increase in
the photosynthetic rate leads consistently to an increase in the tree productivity
in terrestrial ecosystems, although in the long term, down-regulation associated
with the nutrient or water availability may influence the magnitude considerably
(for a review, see Medlyn et al. 2011). If no down-regulation is considered, model
17 Climate Feedbacks Linking the Increasing Atmospheric CO2 . . . 493

estimates predict consistent increases in net primary production (NPP), and even
more so if the interaction with temperature and prolonged growing season is
taken into account. These predictions are supported by the results of free-air CO2
enrichment (FACE) studies (Norby et al. 2005). Thus, although elevated CO2
concentrations may or may not affect the rate of monoterpene emission per unit
foliage mass, the simultaneous effects on the LAI and productivity (in this paper
we use gross primary production, GPP), combined with the effects of increasing
temperature on biosynthesis and volatilization in the short term, and on growing
season length in the long term, are predicted to increase isoprene and monoterpene
emission rates in large regions over the Northern Hemisphere.
Once emitted to the atmosphere, BVOCs participate in atmospheric oxidation
processes initiated by OH and NO3 radicals, ozone and by some yet poorly
quantified oxidants such as stabilised Criegee intermediates (Mauldin et al. 2012).
Some of the resulting oxidation products condense on the pre-existing aerosol
particles increasing their size, and some are able to form the very smallest aerosol
particles formed by atmospheric nucleation. This process, due to the strong size-
dependence of the aerosol scattering coefficient, is expected to lead to an increased
fraction of diffuse solar radiation. Increased diffuse radiation in turn enhances
the plant photosynthesis by providing more light into shaded areas in forest
canopies. This effect of the increased fraction of diffuse to total solar radiation on
photosynthesis has been studied by Mercado et al. (2009) on a global scale using a
global climate and land-use model. This will cause a positive feedback mechanism
between increasing CO2 concentrations and biogenic activity.
Over the boreal forest environment, secondary organic aerosols formed from
BVOCs frequently dominate aerosol particle number concentrations (Tunved et al.
2006), affecting notably aerosol light scattering and even more so CCN concentra-
tions (Lihavainen et al. 2009). Globally, atmospheric new-particle formation and
growth associated with sulphur and BVOC emissions has been suggested to give a
large contribution to the CCN budget (Spracklen et al. 2008; Merikanto et al. 2009;
Pierce and Adams 2009; Yu and Luo 2009; Kerminen et al. 2012), which causes a
major uncertainty in the present-day indirect climate forcing estimates (Wang and
Penner 2009; Kazil et al. 2010; Makkonen et al. 2012a, b). In the future, when
primary aerosol particle concentrations associated with anthropogenic activities are
projected to decline, the climatic role of natural aerosols associated with BVOC
emissions is probably even larger than today (Makkonen et al. 2012a).

17.2 Methods to Analyse the Significance of Feedback Loops

17.2.1 GPP Loop in Boreal Forests

The field data used in this study have been measured during the years 1996–2011
at the University of Helsinki SMEAR II station in Hyytiälä, southern Finland
(61o 510 N, 24o 170 E, 181 m above sea level) (see Fig. 17.2 for a view of the
494 M. Kulmala et al.

Fig. 17.2 A view from the measurement mast of the Hyytiälä SMEAR II station. The station
is located in a Scots pine (Pinus sylvestris) dominated 50-year-old forest. Basic meteorological
parameters such as temperature, relative humidity and wind speed are continuously measured at six
levels of the station’s 74 m tall mast, as well as concentrations of trace gases such as SO2 , CO2 , CO
and O3 . Solar radiation is measured with pyranometers (Middleton Solar and Delta-T Devices Ltd).
Aerosol size distributions are measured in the mobility size range of 3–1,000 nm (3–500 nm until
December 2004) using a twin-DMPS setup (Differential Mobility Particle Sizer; Aalto et al. 2001).
Larger particles are measured with an Aerodynamic Particle Sizer (APS). Enclosure measurements
are performed for gas-exchange (photosynthesis, transpiration and BVOC emissions) at shoot level
(For additional details see Hari and Kulmala 2005)

measurement site). At the SMEAR II station, continuous and comprehensive mea-

surements on the exchange processes between the atmosphere and land-ecosystem
are performed. The SMEAR II station environment represents a typical boreal
coniferous Scots pine (Pinus sylvestris) dominated forest. Further details of the
station and the measurements are given by Hari and Kulmala (2005).
In order to characterize the effect of secondary organic aerosol production on the
number concentration and size distribution of the aerosol population, we calculated
from the Differential Mobility Particle Sizer (DMPS, Fig. 17.2 for the description
of experimental setup) data the condensation sink (CS). The condensation sink
describes the aerosol particles’ ability to remove vapour molecules from air, and
it is closely linked to the total surface area of the particles and can therefore be used
as a proxy for the light scattering properties of the aerosol population. The value of
CS is calculated from the aerosol number size distributions according to (see e.g.,
Kulmala et al. 2012):
X    
CS D 4D ˇm dp dp N dp ; (17.1)
17 Climate Feedbacks Linking the Increasing Atmospheric CO2 . . . 495

where D is the vapour diffusion coefficient of the condensing vapour (typically

assumed to be sulphuric acid when calculating the value of CS), dp is the
particle diameter, ˇ m (dp ) is the Fuchs-Sutugin transition-regime correction factor
for particles of given size, and N(dp ) is the number concentration of particles of
size dp . The summation in Eq. 17.1 is carried over all the size bins of the measured
aerosol size distribution.
To quantify the GPP feedback loop in a consistent way, it is crucial to look at all
components of the loop at a certain temperature and radiation window. Without this
approach, it would be impossible to separate the effects of CO2 increase from the
effects of solar radiation and temperature. Actually, the GPP feedback loop could
also be called clear-sky feedback loop. Here all the calculation steps are based on
measured data.
The initial driving force in the feedback loop is the increase in atmospheric
CO2 concentration, which enhances photosynthesis, i.e., the forest gross primary
production, GPP. The value of GPP is obtained as the difference between the total
ecosystem respiration (TER) and net ecosystem exchange (NEE) of CO2 :

GPP D TER  NEE: (17.2)

Here, NEE was measured with the eddy covariance technique, i.e., has a negative
sign for net carbon uptake by ecosystem (Markkanen et al. 2001; Suni et al. 2003),
whereas TER was modelled on the basis of nighttime NEE measurements (Suni
et al. 2003; Kulmala et al. 2004). This gives the amount of chemical energy fixed by
the vegetation.
In order to obtain as reliable trend in the atmospheric CO2 concentration as
possible, we utilised the measurements made at the Global Atmosphere Watch
station at Mauna Loa, Hawaii. These measurements should represent the global CO2
values in the northern hemisphere as the Mauna Loa station is situated far away from
any local sources of CO2 .
Since BVOC emissions depend on both GPP and temperature, we divided all the
studied data into 5-degree temperature bins and considered each season separately.
The diffuse radiation fraction is also one component in our feedback loop, and to
separate out the effect of cloudiness in the other steps, we divided the measurement
days into cloudy and cloud-free conditions according to the brightness parameter, P,
defined as the daily ratio of the summed global radiation to the theoretical radiation
sum. The theoretical radiation is calculated based on the elevation angle of the sun
and the latitude of the measurement site, and it describes the maximum amount of
solar radiation that can be received in totally cloud-free conditions. The calculation
of the brightness parameter has been explained in more detail by Kulmala et al.
(2010). As a threshold value for a day to be classified as cloudy we used P < 0.3 and
for cloud-free days we used P > 0.6. These values are derived from comparisons of
the brightness parameter to cloudiness estimated from satellite images (Sogacheva
et al. 2008).
Here, the following five connections in the GPP loop (Fig. 17.1) will be
investigated based on the field measurements: (1) the effect of increased CO2
496 M. Kulmala et al.

concentration on the forests’ photosynthesis and gross primary production (GPP)

(the measurement data were taken from the period 1996–2011), (2) the effect of
the changing GPP on the BVOC emissions, more specifically, on monoterpene
concentrations (period 2006–2011), (3) the effect of the changes in monoterpene
concentrations on the SOA concentration in terms of the increase in the value of CS
(period 2006–2011), (4) the connection between CS and diffuse radiation fraction of
the global radiation (period 2000–2009), and (5) the connection between the diffuse
radiation fraction and GPP (period 2000–2009).

17.2.2 Analysing the Temperature-Related Loop

with Modelling

Modelling the feedback loop associated with the ambient temperature increase relies
on several poorly quantified and often very crudely parameterized relationships.
Generally, the BVOC source is prescribed in global aerosol models. With a few
exceptions, global models include the emission data from Guenther et al. (1995,
2006, 2012; Guenther 2013), although optional algorithms and emission inventories
are available (Ashworth et al. 2013; Grote et al. 2013; Niinemets et al. 2013).
However, the simulated aerosol distribution could be rather sensitive to spatial and
temporal differences in BVOC emission fields. When using a climate model, the
simulated climate most likely differs from the climate data used to obtain the results
by Guenther et al. (1995). Recently, the BVOC emission parameterizations have
been implemented to global aerosol models, allowing one to simulate the BVOC-
aerosol-climate feedback interactively (e.g., O’Donnell et al. 2011).
In most global aerosol models, secondary organic aerosol formation is treated
simply as an emission of primary particles. This “primary-SOA” approach dis-
regards all the dynamics and chemistry related to SOA formation, and assumes
that a certain amount of biogenic precursor immediately forms a certain number
of particles, (e.g., Stier et al. 2005). When taking into account the dynamics
of SOA formation, two approaches are generally used: the kinetic one and the
thermodynamic one (Riipinen et al. 2011). The kinetic approach assumes that low-
volatile organic vapours condense irreversibly on the existing particle population,
usually according to the available aerosol surface area, whereas the thermodynamic
approaches assumes equilibrium in the organic vapour concentration between the
gas and aerosol phases. Despite the huge number of different organic compounds
in the atmosphere, their properties are usually lumped together and described with
one or two components. Recently, implementations of the volatility basis set (e.g.,
Donahue et al. 2011) capable of dealing with a larger number of organic vapours
have been introduced into global aerosol modelling frameworks, yet this approach
is computationally too expensive for climate simulations.
The connections between BVOC emissions, SOA formation and CCN concen-
trations are extremely sensitive to the assumptions made on new-particle formation,
primary aerosol size distribution and SOA formation (e.g., Pierce and Adams 2009;
17 Climate Feedbacks Linking the Increasing Atmospheric CO2 . . . 497

Spracklen et al. 2010). In the “primary-SOA” approach, the BVOC oxidation

products do not provide growth for sub-CCN particles, and the CCN-increasing
ability of BVOC emission depends on the assumed size distribution of the primary-
SOA particles and other vapours available for particle growth (e.g., sulphuric acid).
With the kinetic or thermodynamic approaches, the effect of BVOCs on the particle
size distribution depends on the assumptions made on gas-particle partitioning. Pure
thermodynamic partitioning might lead to an underestimation of the condensational
flux to the smallest particles, leading to less growth for newly formed particles and
increased particle sink.
Here, we used the global climate model ECHAM5-HAM (Stier et al. 2005) to
simulate aerosol concentrations, cloud properties and total aerosol forcing with
present-day (year 2000) and future (year 2100) emissions. A detailed description
of the model implementation applied here, including the treatment of BVOC
emissions, new particle formation, SOA formation and aerosol-cloud interaction,
can be found in Makkonen et al. (2012a) and will not be repeated here.
Similar to most other global models of aerosol formation, particulate and gaseous
emissions are implemented in the lowest model level, close to vegetation surface,
in ECHAM5-HAM. The exception is wildfire emissions, which are assumed to
rise higher up in the atmosphere. Sulphate emissions from industry and power
generation are assumed to be injected a bit higher in the atmosphere, around 100–
300 m. This means that, on average, the main sinks of condensable vapours (CS)
and newly-formed particles (coagulation sink) are highest in the lowest model level
with a sharp decrease with rising altitude. Following the decrease in condensation
sink, the highest nucleation rates are usually found in the second-lowest model level.
Near the surface, a larger fraction of the organic vapours formed by BVOC oxidation
condense on pre-existing particles, increasing the condensation and coagulation
sinks and decreasing new-particle formation rates. As a result, increased BVOC
emissions may lead to decreased total particle number concentrations in the near-
surface air. However, higher up in the atmosphere, where the influences of BVOCs
on condensation and coagulation sinks are less effective, the increased particle
growth rates caused by BVOCs act to increase the survival probability of nucleated
particles.

17.3 Significance of the Feedback Loops

17.3.1 Quantitative Experimental Evidence on the Feedback

Loop Associated with GPP

In the first step, which initiates the feedback loop, we studied the effect of increasing
atmospheric concentration of CO2 on the biogenic activity of the forest at the
Hyytiälä SMEAR II station. This effect is called CO2 fertilization. As a direct
indicator of this biogenic activity we use the gross primary production (GPP)
498 M. Kulmala et al.

Fig. 17.3 Annual average

gross primary production as a 15
function of air CO2 mole

GPP (μmol m−2 s−1)

fraction in the Hyytiälä P. 14
sylvestris dominated boreal
forest (Fig. 17.2 for the site).
The data are taken on clear 13
days (brightness parameter
larger than 0.6) from months
May-August in the 12
temperature range 18–23 ı C.
The solid line shows the 11
linear least-squares fit to the
data (r D 0.74, P < 0.002) 360 370 380 390 400
CO2 concentration (μmol mol−1)

which describes the carbon uptake from the atmosphere into biosphere by the
photosynthetic activity of the plants. The annual average GPP measured at the
SMEAR II station by eddy covariance technique correlated positively with the
atmospheric CO2 concentration (we used the CO2 measurement data from Mauna
Loa representing the northern hemisphere average conditions). The correlation of
annual average GPP with the atmospheric CO2 concentration during the months
May-August is shown in Fig. 17.3. The increase rate is on the order of 1 % per ppm
of CO2 concentration increase.
A significant correlation of the volatile organic vapour concentrations with the
gross primary production was observed in the Hyytiälä Scots pine (P. sylvestris)
dominated forest across the whole study period of 15 years (Fig. 17.4a). We used
here the measured monoterpene concentrations, which are known precursors for
condensing organic vapours. The highest observed monoterpene concentrations
seemed to increase approximately linearly with increasing GPP, whereas at high
GPP levels, also low monoterpene concentrations could be observed. Increasing
monoterpene concentrations led to increased values of condensation sink, as shown
in Fig. 17.4b. This is caused by the monoterpene oxidation products condensing on
pre-existing particles and increasing their size, and hence their surface area which is
related to the CS. Increased concentrations of oxidized BVOC can also enhance the
growth of particles from nucleation events, which leads to increase in the number
concentration of particles in the Aitken and even in accumulation modes. Especially,
the accumulation mode particles larger than 100 nm in diameter contribute strongly
to the condensation sink. The accumulation mode particles are also large enough to
scatter sunlight. This was seen in our observations as positive correlation between
the CS and the diffuse fraction of global radiation (Fig. 17.5a). Finally, to close the
observational feedback loop, we plotted the daily average GPP against the diffuse
radiation fraction in Fig. 17.5b. Although there were also high values of GPP at
low values of Rdiff /Rglob , the highest values of GPP and no low values of GPP were
observed when the ratio Rdiff /Rglob was high. In all the steps of the feedback loop,
the results shown in Figs. 17.4 and 17.5 indicate a statistically significant positive
correlation.
17 Climate Feedbacks Linking the Increasing Atmospheric CO2 . . . 499

Fig. 17.4 Monoterpene ambient air concentration as a function of gross primary production (a)
and the condensation sink (Eq. 17.1) as a function of the monoterpene concentration (b) in the
Hyytiälä P. sylvestris dominated boreal forest. The number of data points (daily averages during
months May–August in the years 2006–2011) is 78 for (a) and 76 for (b). The data were selected
such that the temperature was in the range 18–23 ı C and the brightness parameter larger than
0.6 (clear days). Monoterpene concentration was measured with a proton-transfer reaction mass
spectrometer (PTR MS). The solid lines show the linear least-squares fits to the data (r D 0.28,
P < 0.02 for (a) and r D 0.27, P < 0.02 for (b))

Fig. 17.5 Diffuse radiation fraction of the observed global radiation (Rdiff /Rglob ) as a function
of the condensation sink (a) and gross primary production as a function of Rdiff /Rglob (b) in the
Hyytiälä P. sylvestris dominated boreal forest. The number of data points (daily averages during
months May–August in the years 2000–2009) is 224 for (a) and 230 for (b). The data selection as
in Fig. 17.4. The solid lines provide the linear least-squares fits to the data (r D 0.39, P < 0.001 for
(a) and r D 0.28, P < 0.001 for (b))
500 M. Kulmala et al.

a b

cm−3

< −50 −20 −10 0 10 20 50 >

Fig. 17.6 Simulated global absolute increases in the concentration (cm3 ) of cloud condensation
nuclei at 0.2 % supersaturation, CCN(0.2 %), due to doubling of BVOC emissions and with
anthropogenic emissions of the year 2000 (a) and 2100 (b)

Short-term drivers may decouple the link between CO2 and BVOC emissions,
especially isoprene (see e.g., Arneth et al. 2007, 2011), but these processes are not
taken into account here. Water limitation can further override the inhibitory effect
of elevated CO2 , leading to increased isoprenoid emissions in a climate change
scenario with warmer and drier climate (Pegoraro et al. 2005; Sun et al. 2012). As
discussed by Arneth et al. (2011), the overall direction and magnitude of emission
change will strongly depend on the vegetation changes, relative importance of
the short-term and longer-term factors, and the potential physiological acclimation
responses resulting in gradual and continuous increase in CO2 .

17.3.2 The Feedback Loop Associated with Temperature

As a demonstration of the potential strength of the feedback loop associated

with future temperature increases, we made model simulations with standard and
doubled global BVOC emissions. Since the connection between BVOC emissions
and resulting changes in CCN concentrations and cloud properties are expected
to depend strongly on anthropogenic emissions, the simulations were made with
both present-day (year 2000) and anticipated future (year 2100) anthropogenic
aerosol and their precursor emissions. Yet another simulation was carried out with
the year 1750 emissions in order to calculate the radiative forcing. In our model
implementation, doubling monoterpene emissions from 127 to 254 Tg per year
increased the annual SOA formation from 19 to 38 Tg of SOA, which is still
in the low end of SOA formation estimates (Carslaw et al. 2010). Maps of CCN
concentration changes in different model runs are shown in Figs. 17.6 and 17.7 and
summarized in Table 17.1 and Fig. 17.8.
17 Climate Feedbacks Linking the Increasing Atmospheric CO2 . . . 501

a b

< −50 −30 −10 0 10 30 50 >

Fig. 17.7 Modelled relative increases (%) in CCN(0.2 %) concentrations due to doubling of
BVOC emissions and with anthropogenic emissions of the year 2000 (a) and 2100 (b)

Table 17.1 Global average surface-level concentration (cm3 ) of cloud condensation

nuclei at 0.2 % supersaturation, CCN(0.2 %), over land and ocean areas in years 2000 and
2100
Decrease between
Year 2000 Year 2100 years 2000 and 2100
Land Ocean Land Ocean Land Ocean
1  BVOC 217 60 89 38 60 % 36 %
2  BVOC 226 62 93 41 59 % 35 %
Increase due to doubled C4 % C3 % C5 % C6 %
BVOC emission
Bottom row shows the increase in CCN(0.2 %) concentration due to doubling of BVOC
emissions. The right columns show the decrease in CCN(0.2 %) concentration due to
changes in anthropogenic emissions between years 2000 and 2100

With the exception of Central and Northern Africa, doubling of BVOC emission
with present-day (year 2000) anthropogenic emissions increased CCN concentra-
tions above the continental areas (Fig. 17.6a). The strength of this increase was
related to the number concentration of sub-CCN sized particles in each location:
local hotspots of CCN increases can be seen in Europe, North America and South
Africa, which all have larger numbers of sub-CCN size anthropogenic particles
available for growth. To the contrary, tropical regions in Africa and South America
showed smaller concentrations of sub-CCN particles. Although BVOC emissions
are high in the tropics, absolute increases in CCN concentrations were modest there
compared with regions affected by anthropogenic emissions. Over areas dominated
by sea-salt or dust particles, a major portion of condensable organic vapours was
taken up by these relatively large particles, leading to a slower growth of newly-
formed particles. AeroCom recommendations (Dentener et al. 2006) followed by
many global aerosol models suggest a rather large primary particle emission size
502 M. Kulmala et al.

95

Doubling BVOC
Cloud drouplet number concentration (cm−3)
90 pogenic emissions
No change in anthro

85

80

R
ed
uc o c
D

tio
ou

n
75

N
bl

in
in

an
g

ha
BV

th
ng

ro
70 O

po
C

in

ge
BV

ni
c
O
65

em
C
em

is
si
iss

on
s
io
60

n
55

50
Year 2000 Year 2100

Fig. 17.8 Predicted change in global cloud droplet number concentration (CDNC, cm3 ) due to
changes in anthropogenic and biogenic emissions

(median diameter 80 nm) from wild-fires. These particles are already near cloud
droplet activation sizes at the time of emission, which reduces the sensitivity to
BVOC emission in areas dominated by wild-fires.
The cooling effect of an increased cloud albedo is higher when the contrast
between land and cloud albedo is higher. Above a snow-covered landscape, a large
portion of radiation would be reflected back to space even without clouds, leaving
less room for the first aerosol indirect effect. On the other hand, above a dark forests
or ocean, the difference between cloud and land albedo can be very large. Even
when assuming the SOA formation from BVOCs to take place over the continents
only, some fraction of particles affected by the SOA formation will be transported
to atmosphere over oceans. In our simulations, doubling of continental BVOC
emissions increased CCN concentrations by 4 % over land and 3 % over ocean areas
(Table 17.1). The doubling of BVOC emissions with present-day anthropogenic
emission levels leads to a 3 % increase in global CDNC in our simulations, which
modified both cloud albedo and lifetime causing the global cloud radiative effect
of about 0.2 W m2 . This suggests that the cooling potential associated with the
feedback loop connecting BVOC emissions to increased ambient temperatures is
definitely non-negligible.
As discussed before, the indirect effects of BVOCs depend on the existing
population of sub-CCN particles. In many models, the continental sub-CCN
sized particles are mainly of anthropogenic origin. Strong emission reductions of
anthropogenic primary particles and aerosol precursors are predicted throughout
the twenty-first century (Lamarque et al. 2011). The reductions in particle number
concentration will hence decrease the CCN formation from BVOCs. However, the
17 Climate Feedbacks Linking the Increasing Atmospheric CO2 . . . 503

overall decrease in the CCN concentrations might lead to an increased susceptibility

of cloud albedo to CCN concentrations. Figure 17.6b shows increases in CCN
concentrations due to the doubling of BVOCs when using anthropogenic emissions
for the year 2100. While the absolute global CCN concentration increase of 3 cm3
with the year 2100 anthropogenic emissions is slightly less than with present-day
emissions (4 cm3 ), the relative increase is higher due to overall lower aerosol
concentrations (Fig. 17.7). Also, the land/ocean contrast is changed with present-
day and future anthropogenic emissions (Table 17.1). Due to the reductions in
anthropogenic emissions, CCN concentrations over the oceans are decreased by
36 %, and the relative effect of doubling BVOC emission is increased from 3
to 6 %. This could indicate potential modification in marine cloud properties.
Relative increases in CCN concentrations due to doubling of BVOC emissions are
emphasized at the high latitudes of the Northern Hemisphere (Fig. 17.7).
It is clear that the magnitude of the possible climate feedback via BVOC
emissions is strongly dependent on the evolution of anthropogenic emissions
(Fig. 17.8). In addition to providing seed particles for further growth, human
activities can influence the oxidative capacity of the atmosphere. The SOA formed
from BVOCs with the aid of anthropogenic pollutants can make a large contribution
to the total SOA (Spracklen et al. 2011), which is another example of couplings in
the climate system that should be investigated further.

17.4 Conclusions

We have illustrated and investigated two partly interacting climate feedback loops
connected to terrestrial BVOC emissions, both driven by increased atmospheric
CO2 concentrations. The central point of first of these two loops is the plant gross
primary production (GPP), which is hypothesized to be fed back positively via
BVOC-induced organic aerosol formation and resulting changes in the ratio between
diffuse and global solar radiation. The central point of the second loop is the ambient
temperature which has been hypothesized to be fed back negatively via BVOCs-
induced CCN production and resulting changes in the cloud properties.
The feedback loop associated with GPP was investigated here by analysing
15 years of atmospheric measurement data from a boreal forest site in Hyytiälä,
Finland. A firm positive relation between all drivers forming the individual links in
this feedback cycle was found, providing strong empirical support for the existence
of such loop in a boreal forest environment. In future, we should aim to find out
whether the same feedback loop is active in other terrestrial ecosystems as well and,
using combination of measurement and modelling tools, to quantify the strength of
this feedback.
The feedback loop associated with the ambient temperature increase was inves-
tigated here by using global model simulations. While subject to large uncertainties
due to several simplifying assumptions, our simulations demonstrated that this
second feedback loop may be regionally very important, and non-negligible even
504 M. Kulmala et al.

in the global atmosphere. The overall strength of this feedback is likely to be

increased in the future when anthropogenic emissions of primary aerosols and
aerosol precursor gases are expected to decline.
From the atmospheric chemistry and aerosol system point of view, BVOCs are
crucial both locally and globally because of their important role in both new particle
formation and growth. By participating actively in chemical reactions, including the
atmospheric oxidation capacity, BVOCs connect several feedback loops involving
the biosphere and atmosphere. In order to quantitatively assess the impact of BVOCs
on future biosphere-atmosphere-climate interactions and feedbacks, it is important
to understand in more details the life cycle of BVOCs in the atmosphere.

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Chapter 18
State-of-the-Art of BVOC Research:
What Do We Have and What Have
We Missed? A Synthesis

Ülo Niinemets and Russell K. Monson

Abstract This book summarizes recent advancements in the resolution and quan-
tification of the controls on tree BVOC emissions, including efforts toward synthetic
projections using computer models. Major progress has been achieved in under-
standing the molecular mechanisms of volatile synthesis and emission, the role of
emissions in plant stress tolerance and elicitation of emissions under biotic and
abiotic stresses. Use of this rich source of insight not only allows for improvement
of regional air quality estimations under current climate and atmospheric conditions,
but it also allows for improvements to the models and observations needed to
predict BVOC emissions under future climate and atmospheric conditions. As
our understanding of physiological mechanisms, taxonomic distribution and multi-
trophic interactions in forest ecosystems increases further, we will be able to tackle
some of the large-scale feedback loops between BVOC emissions, plant stress, and
climate that have eluded us for so long.

18.1 Determinants of Diversity of BVOC Emitters

and Emission Diversity

Trees are traditionally considered to be the key BVOC emitters and they make the
greatest contributions to BVOC emissions worldwide. It has been a long-standing
enigma as to why plants emit those BVOCs not involved in herbivore protection,

Ü. Niinemets ()

Department of Plant Physiology, Institute of Agricultural and Environmental Sciences,
Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, Estonia
e-mail: [email protected]
R.K. Monson
School of Natural Resources and the Environment and the Laboratory
for Tree Ring Research, University of Arizona, Tucson, AZ 85721, USA
e-mail: [email protected]

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 509
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8 18,
© Springer ScienceCBusiness Media Dordrecht 2013
510 Ü. Niinemets and R.K. Monson

in particular, isoprene, and light-dependent monoterpenes, and why not all, but only
some species exhibit high rates of constitutive emissions? It is particularly enigmatic
as to why this trait is so clearly associated with woody, principally tree, species.
Studies with the plastidic isoprenoid pathway inhibitor, fosmidomycin, and with
isoprene fumigation (Sharkey and Singsaas 1995; Singsaas et al. 1997; Loreto et al.
2001b), and work with transgenic plants with repressed (Behnke et al. 2007, 2010;
Rosenkranz and Schnitzler 2013) or constitutively increased (Vickers et al. 2009;
Velikova et al. 2011) isoprene emission suggests that isoprene emission increases
tolerance to recurrent cycles of extremely high temperature, drought and oxidative
stress. Similarly, experiments with suppression of constitutive monoterpene emis-
sion by fosmidomycin-feeding followed by monoterpene fumigations (Loreto et al.
1998, 2001a; Delfine et al. 2000; Copolovici et al. 2005; Llusià et al. 2005) have
demonstrated that monoterpenes can fulfil analogous functions in leaves.
Thus, there appears to be an important role for constitutive emissions in abiotic
stress tolerance. The question remains, however, as to why this trait is expressed
in only some tree species. Multiple monoterpene synthase genes are present in all
plant species, and plants that store monoterpenes in their leaves, and emit these
compounds, occur among both woody and herbaceous species. In other species
without storage structures, monoterpene emissions can be triggered (induced or
upregulated) by multiple biotic and abiotic stresses (Loreto and Schnitzler 2010;
Niinemets 2010; Trowbridge and Stoy 2013). Yet, the constitutive emissions of de
novo monoterpene emissions in species without specialized storage are relatively
rare. Obviously, modification in the regulatory elements in the promoter sequence
of given monoterpene synthase genes can change the mode and location of monoter-
pene synthase expression, but information concerning the promoter sequences of
monoterpenes is limited (Rajabi Memari et al. 2013).
Isoprene synthase genes analysed so far have been found to have high se-
quence homology with certain monoterpene synthase genes (Miller et al. 2001;
Sharkey et al. 2005) and even a bifunctional acyclic monoterpene (myrcene)
synthase/isoprene synthase gene has recently been described (Sharkey et al. 2013).
Given the presence of multiple monoterpene synthases, and accordingly, the
potential for multiple origins of isoprene synthase, it is even more surprising why
a trait that improves tolerance to abiotic stress has not evolved in all plant species.
Exposure to heat, drought and oxidative stress occurs broadly across taxonomic
groups. Fineschi et al. (2013) in their chapter analyse recent phylogenetic evidence
(Monson et al. 2013; Sharkey et al. 2013) and suggest that the probability of
occurrence of isoprene emission is strongly driven by climatic conditions and
atmospheric level of CO2 , as well as phylogenetic inertia. The benefits of isoprene
emission are larger when photosynthesis is suppressed due to the combination of
low atmospheric CO2 concentration and stress (thus, increasing the need for non-
photochemical energy quenching), whereas stress amelioration by a molecule that
is volatile provides its greatest benefit when the stress is episodic, not chronic.
Thus, the evolutionary diversification of isoprene emission is particularly favoured
in atmospheres with low [CO2 ] and in relatively humid environments which may
nevertheless occasionally experience extremely high temperatures and water stress
18 State-of-the-Art of BVOC Research: What Do We Have and What Have. . . 511

(Fineschi et al. 2013). In fact, phylogenetic evidence suggests multiple events

of loss and evolution of isoprene emission (Monson et al. 2013). On the other
hand, emission has a cost in terms of photosynthetic carbon loss, especially
under stress, and thus, isoprene emission seems to be confined to plant genera
that occur frequently in habitats such as open, early-successional forests where
photosynthesis is higher due to greater light availability, while emissions of less
volatile monoterpenes are more common among late-successional species that often
tend to grow under and form a dense canopy (Harrison et al. 2013).
There clearly seem to be cost-benefit effects operating on the appearance and
disappearance of isoprene, and light-dependent monoterpene emissions. A second
intriguing question is: what determines the blend of different monoterpenes?
Monoterpene synthase specificity varies, with some synthases capable of producing
a greater diversity of products, and others producing a single product (Degenhardt
et al. 2009; Rajabi Memari et al. 2013). Although the structure of several key
terpene synthases has been characterized (Hyatt et al. 2007; Köksal et al. 2011;
McAndrew et al. 2011; Zhou et al. 2012), there is currently no objective way to
predict terpene synthase properties on the basis of sequence homology and active
site structure (Degenhardt et al. 2009). Even very high sequence and structural
homology does not necessarily imply that the two paralogs have the same or even
similar terpene product profiles. From an evolutionary perspective, this implies that
terpene profiles can be altered with minimum changes in sequence (Kampranis et al.
2007), making the terpene emission profile very ‘plastic’ in terms of evolutionary
diversification. Evidence that different monoterpenes can differently alter plant
abiotic stress tolerance (Copolovici et al. 2005) further underscores the importance
of understanding the role of emission blends.
From the plant-to-plant and plant-to-insect communication perspective, the role
of differences in induced terpene emission blends has been known for a long time
(Dicke and Bruin 2001; Dicke and Baldwin 2010; Trowbridge and Stoy 2013
for reviews), and thus, the capacity for changes in the emission profiles with
minor changes in genome may be a common evolutionary outcome, and in fact,
a necessity to cope with continual changes in herbivory pressure. Although we
do not generally think about constitutive emissions in the infochemical context,
the transgenic introduction of constitutive isoprene emissions into an otherwise
non-emitting plant resulted in greater herbivory resistance (Loivamäki et al. 2008),
suggesting a function for isoprene emissions beyond that of enhancing abiotic stress
tolerance. Given that the emissions of many constitutive BVOCs are enhanced under
mild stress (Niinemets 2010) and that priming by mild stress often results in reduced
host quality, constitutive BVOCs might serve further functions we have not yet
identified. There is no reason to believe that the evolutionary origins of traits, such
as terpene emissions, are linked to isolated, individual effects on fitness. Synergy
among adaptive benefits is possible, and should be explored in future studies of
fitness in transgenic plants with variable levels and types of BVOC emissions.
So far, genetically engineered plants with introduced isoprene (Sasaki et al. 2007;
Vickers et al. 2009; Velikova et al. 2011) or mono- and sesquiterpene (Wu et al.
2006) emissions were only available for herb model systems. In this book, for the
512 Ü. Niinemets and R.K. Monson

first time, transgenic birch (Betula pendula) lines are introduced (Rosenkranz and
Schnitzler 2013), providing an exciting new tree model system to test the impact
of constitutive introduction of isoprene emission on plant abiotic and biotic stress
resistance.

18.2 Molecular, Pathway and Genetic Controls on Emissions

Phenomenological evidence of light and temperature controls on isoprene emission

(Sanadze and Kalandadze 1966; Sanadze 1969; Tingey et al. 1981, 1987 Evans
et al. 1982; Monson and Fall 1989; Monson et al. 1991) constituted the indirect
support that isoprene must be formed enzymatically. This was proven with the
discovery of isoprene synthase (Silver and Fall 1991), but at that time it was
believed that isoprene emission occurred through the use of substrates generated
in the mevalonate-dependent pathway in the cell cytosol, and thus, it was difficult to
explain why isoprene emission was so tightly linked to photosynthesis. Especially
to the point that the isotopic label from CO2 was detected in emitted isoprene
molecules within only a few minutes after the start of labelling (Sanadze et al. 1972;
Delwiche and Sharkey 1993). This enigma was not resolved until it became widely
known that 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate
(MEP/DOXP) pathway located in the chloroplasts is responsible for the bulk of
volatile isoprenoids emitted (Lichtenthaler 1999; Zeidler and Lichtenthaler 2001).
Discovery of the MEP/DOXP pathway has catalyzed rapid development in
resolving the molecular and pathway controls on isoprene emission (Li and Sharkey
2013b; Monson 2013). The key issue in understanding these controls has been
whether the emission is limited by isoprene synthase activity or by the concentration
of its immediate substrate, dimethylallyl diphosphate (DMADP), in chloroplasts. As
isoprene synthase requires Mg2C ions as an enzymatic co-factor, as do all known
type I terpene synthases with the active site located in the ’-domain of the protein
(Aaron and Christianson 2010; Köksal et al. 2010; Rajabi Memari et al. 2013), it has
been suggested that light-dependent changes in Mg2C are responsible for the light
response of isoprene emission (Silver and Fall 1991). On the other hand, in early
studies there was evidence of correlation of isoprene emission rate with cellular ATP
and carbon intermediate levels (Loreto and Sharkey 1993). However, researchers
faced a significant challenge in resolving the role of these alternative hypotheses in
regulating the light-dependence of emission because of difficulties in estimating the
size of the DMADP pool, and especially that of the chloroplastic pool. Methods for
estimating the DMADP pool from cellular extracts were developed, but these did
not have the potential to completely separate those fractions of the pool occurring
in the cytosol versus the chloroplast (Fisher et al. 2001; Rosenstiel et al. 2002).
Development of methods based on 13 C-labelling of the DMADP pool (Loreto
et al. 2004) and the kinetics of post-illumination isoprene emission (Rasulov et al.
2009a) provided conclusive evidence that DMADP pool size is controlled by light
availability (Loreto et al. 2004; Rasulov et al. 2009a), while isoprene synthase
18 State-of-the-Art of BVOC Research: What Do We Have and What Have. . . 513

activity does not change during light–dark transients, at least during the initial
tens of minutes to a few hours after changing the light level (Rasulov et al.
2009b). Recent work has suggested that the key factor determining the chloroplastic
DMADP pool size in the face of changes in the photosynthetic photon flux density is
either ATP availability or the activities of several MEP pathway reductive enzymes
that can accept electrons directly from the photosynthetic electron transport chain
(Rasulov et al. 2011; Li and Sharkey 2013a). This does not mean that the availability
of ATP controls the emissions per se as the overall requirement for ATP of
isoprene emission is small compared with photosynthesis, but rather it suggests
that a high effective Km for ATP exists within the MEP pathway. This, combined
with the previously-discovered exceptionally high Km for DMADP exhibited by
extracted isoprene synthase as well as in vivo (Schnitzler et al. 2005; Rasulov et al.
2009a; Li and Sharkey 2013b), implies that light-dependent changes in DMADP
are immediately reflected in alterations in isoprene emission rates. Despite major
progress in understanding these chloroplastic relations as controls over the light-
dependency of isoprene emissions, there is also a great need for studies of isolated
protoplasts and chloroplasts, as well as targeted transgenic manipulation, to validate
some of the hypothesized metabolic connections.
Control of isoprene emission rate by intercellular CO2 , a response exhibiting an
optimum at intercellular CO2 concentrations between 120 and 200 mol mol1 and
declining towards lower and higher CO2 concentrations, has also been attributed
to changes in chloroplastic DMADP concentration (Rasulov et al. 2009b; Monson
2013). There is, however, debate over the mechanism that links changes in CO2
concentration to alterations in the chloroplastic DMADP pool size (Monson 2013).
In one set of studies, alterations in the activity of the enzyme phosphoenol pyruvate
(PEP) carboxylase in isoprene emitting leaves have been shown to result in recipro-
cal changes in the isoprene emission rate (Rosenstiel et al. 2003, 2004). Given that
PEP carboxylase is a cytosolic enzyme that assimilates bicarbonate (in equilibrium
with cytosolic CO2 concentration), these results supported the hypothesis that the
channeling of pyruvate substrate via phosphoenol pyruvate from the cytosol to
the chloroplast controls the chloroplastic DMADP concentration, and therefore
controls the response of isoprene emissions to changes in CO2 concentration. In
the chapter by Li and Sharkey (2013b), a new hypothesis has been proposed that
links the import of PEP into the chloroplast to limitations in inorganic phosphate
(Pi ) availability, which is altered by the balance between the rates of photosynthesis
as influenced by CO2 concentration and sucrose biosynthesis in the cytosol. There
is also evidence that changes in the intercellular CO2 concentration can influence
chloroplastic ATP concentration, once again due to changes in the rate of ATP
turnover as photosynthetic rate and use of the immediate products of photosynthesis
change with changing CO2 concentration (Kiirats et al. 2009). This, in turn, can
lead to changes in DMADP pool size and isoprene emission (Rasulov et al. 2009b,
2011). Thus, while we have an ample set of hypotheses ‘on the table in front of us’,
there is much work to do to better resolve the precise mechanisms underlying the
CO2 sensitivity of isoprene emission in leaves.
514 Ü. Niinemets and R.K. Monson

Of course, it is not necessary to attribute all control to dynamics in the chloroplast

DMADP concentration, but isoprene synthase activity can itself be an important
control over the emission rate. There is a large variation in isoprene emission
rates among leaves that have developed under different environmental conditions
and in leaves of different age, reflecting differences in the amount of isoprene
synthase (Harley et al. 1996; Sharkey et al. 1999; Hanson and Sharkey 2001;
Wiberley et al. 2005, 2008; Niinemets et al. 2010a). This level of control has been
defined as “genetic control” to reflect its underlying mechanisms (Monson 2013).
Isoprene synthase promoter sequences have been described and multiple regulatory
elements, including circadian control elements and temperature-dependent elements
have been identified (Loivamäki et al. 2007; Cinege et al. 2009; Rosenkranz and
Schnitzler 2013). This level of control is responsible for changes in isoprene
synthase activity in response to past temperature, light and CO2 conditions and we
suggest that future work in resolving controls in isoprene emission should clearly
distinguish between metabolic (changes in DMADP substrate) and genetic (changes
in the expression of isoprene synthase genes) controls.
Finally, it is important to recognize that the controls over the emission of BVOCs
can also occur beyond the processes of cellular metabolism and genetic expression.
Physico-chemical properties of emitted volatiles themselves can alter the emission
flux (Niinemets et al. 2004; Harley 2013). Stomata constitute the ultimate ‘valve’
for diffusion of BVOCs from leaves, and traditionally, based on the analogy to
diffusion of CO2 and water vapour in photosynthesis, stomata were considered to
control BVOC emission flux (Harley 2013). However, no significant control by
changes in stomatal conductance on isoprene and non-oxygenated monoterpene
emissions has been observed (Sharkey 1991; Fall and Monson 1992; Loreto et al.
1996) while methanol emissions (Nemecek-Marshall et al. 1995; Harley et al. 2007)
and oxygenated monoterpene emissions (Niinemets et al. 2002) were characterized
by strong stomatal limitations. These discrepancies among the various compounds
were not understood until dynamic models that considered the physico-chemical
properties of volatiles were developed (Niinemets and Reichstein 2003; Harley et al.
2007). These models suggested that stomatal control of BVOC emissions was not
possible in the steady-state conditions, because the rise in the partial pressure of
the volatile compound in leaf intercellular air space, and accordingly the driving
force for diffusion, always compensated for reductions in stomatal conductance
(Niinemets and Reichstein 2003; Harley et al. 2007; Harley 2013). However, the
question is how fast the diffusion gradient changes, i.e., how quickly steady-state
conditions are established after changes in stomatal openness. Stomata have the
potential to influence the rate of emission in transient, non-steady-state conditions,
especially, for compounds with high solubility in the aqueous phase of the leaf,
such as for methanol and oxygenated monoterpenes, while for compounds such as
isoprene and non-oxygenated monoterpenes, with low solubility, stomata can have
minimal influence on observed emission rates.
Once the importance of compound solubility, as a factor in determining the
potential for stomatal limitations had been recognized, it was also recognized
that existence of a certain capacity for non-specific storage of compounds in leaf
18 State-of-the-Art of BVOC Research: What Do We Have and What Have. . . 515

aqueous and lipid phases also implies that even in species without specialized
storage structures, there are time-lags between compound synthesis and emission,
complicating emission responses to environmental drivers (Noe et al. 2006; Harley
2013). On the other hand, constitutively non-emitting species can adsorb lipid-
soluble volatiles released by neighboring plants (Noe et al. 2008; Himanen et al.
2010) and this can lead to greater fitness in terms of reduced herbivory pressure or
improved thermal tolerance or priming for induced defences (Copolovici et al. 2005;
Llusià et al. 2005; Frost et al. 2008; Himanen et al. 2010). Thus, physico-chemical
characteristics of emitted compounds can have major effects on the species interac-
tions in vegetation consisting of constitutive and non-constitutive emitters (Baldwin
et al. 2006). This is an area that clearly needs high priority in future studies testing
for the effects of plant-to-plant communications, or lack thereof in field experiments
with transgenic models with modified emission profiles of the targeted compounds.

18.3 Stress and Emissions: From Constitutive

to Induced Emissions

Being sessile in their life habits, plants have had to evolve multiple chemical
mechanisms to signal other parts of the same organism, as well as other organisms,
about the nature of an encountered stress and its magnitude; plants cannot simply
move to a more favorable environment to avoid the stress. This constraint has
led to a diversity of constitutive and induced capabilities in the production and
emission of BVOCs and their use as ecological signalling cues (Possell and
Loreto 2013; Trowbridge and Stoy 2013). Apart from the role of constitutive
emissions in abiotic stress resistance and possible involvement in biotic interactions
(Sect. 18.1), environmental and biotic stresses are known to induce emissions of
a series of different volatile classes including emissions of oxygenated volatiles
such as methanol and volatile products of lipoxygenase pathway such as various
C6 aldehydes, also called green leaf volatiles, and induced emissions of mono-,
sesqui- and homoterpenes (Beauchamp et al. 2005; Steindel et al. 2005; Loreto
et al. 2006; von Dahl et al. 2006; Copolovici and Niinemets 2010; Holopainen and
Gershenzon 2010; Loreto and Schnitzler 2010; Toome et al. 2010; Copolovici et al.
2012), reflecting the convergence of stress signalling pathways, likely at the level
of stress-driven formation of reactive oxygen species (ROS) (Fujita et al. 2006).
While the same BVOC classes are induced by many stresses, the volatile blends
are often different for different stresses, and typically carry strong species-specific
signatures (Dicke et al. 2009; Loreto and Schnitzler 2010). These variations in the
blend of elicited volatiles have major impacts on plant-insect interactions (Pichersky
and Gershenzon 2002; Bruce et al. 2005; Unsicker et al. 2009), and plant-plant
interactions (Baldwin et al. 2006; Kessler et al. 2006). Thus, clearly there is a need
to gaining insight into the “plant talk”, but currently our learning extends to only
a few herbaceous model systems, and even in these systems, our understanding is
highly superficial.
516 Ü. Niinemets and R.K. Monson

In addition to ubiquitous stress-elicited volatiles, specific volatile emissions are

characteristic to some stresses such as root-zone anoxia characterized by high
emissions of ethanol and acetaldehyde (Kreuzwieser and Rennenberg 2013). These
emissions characterize shifts in root zone heterotrophic metabolism from glycolysis
with Krebs cycle to glycolytic fermentation and ethanol oxidation to acetaldehyde
in leaves. There has been major progress in understanding the controls on ethanol
and acetaldehyde emissions by the rate of transport of ethanol from roots to
leaves and ethanol dehydrogenase activity (Kreuzwieser et al. 2000, 2001, 2004).
However, it is still difficult to predict the magnitude of flood-driven emissions,
especially under natural conditions (e.g., Bracho Nunez et al. 2009). The emissions
are surprisingly poorly linked to species-specific flood tolerance, and specifics
of flood treatment such as duration of flooding (Kreuzwieser and Rennenberg
2013). We may gain greater insight into differential responses of species to flood
treatments through characterization of actual oxygen availability in the root zone
and assessment of species-specific morpho-physiological traits that improve oxygen
availability in root tissues, such as the capacity for aerenchyma and adventitious
root formation. Broader taxonomic characterization of these traits and their efficacy
toward ameliorating anoxic stress might allow us to predict anoxic stress induced
BVOC emissions over large areas potentially impacted by flooding.
In general, there is a reverse correlation between the magnitude of constitutive
and induced emissions under stress (Loreto and Schnitzler 2010; Niinemets 2010;
Possell and Loreto 2013). However, this response is typically observed after severe
stress is reached, while the stress can initially enhance the constitutive isoprene and
monoterpene emissions. Such initial enhancements have been reported for drought,
heat and ozone stress (Sharkey and Loreto 1993; Loreto et al. 1998; Staudt and
Bertin 1998; Pegoraro et al. 2004; Velikova et al. 2005; Calfapietra et al. 2013;
Possell and Loreto 2013). Such enhancement lasts until the stress becomes severe
enough to curb overall photosynthetic and respiratory metabolism. Our estimates of
isoprene biosynthesis rate during abiotic stress, and the true enhancement, however,
may be even higher than we originally estimated. Recent observations have revealed
that a significant fraction of the synthesized isoprene may react with reactive
oxygen species (ROS) inside leaves as part of the metabolic tolerance mechanism
(Jardine et al. 2011). Such internal reactions of constitutive isoprene emission
clearly require more attention in future studies. In fact, ‘overshoots’ of isoprene
emission rates (higher than pre-stress rates) following the relief of stress have been
occasionally observed (Possell and Loreto 2013 for a review), indirectly supporting
the consumption of part of the synthesized isoprene during the stress. In this regard,
the initial upregulation of both constitutive isoprene and monoterpene emissions
by moderately high concentrations of the strong oxidant, ozone, is particularly
interesting (Calfapietra et al. 2013) as this evidence suggests that the rate of consti-
tutive emissions may be modulated at the level of ROS. Ultimately, severe drought,
heat or ozone stresses lead to reductions of constitutive emissions and elicitation
of induced emissions (Calfapietra et al. 2013; Possell and Loreto 2013), but here
the puzzling issue is how the balance between constitutive and induced emissions
is modulated by ROS. Is there a certain threshold beyond which the capacity for
18 State-of-the-Art of BVOC Research: What Do We Have and What Have. . . 517

constitutive BVOCs for ROS detoxification is not enough, triggering elicitation of

stress signalling pathways or is the stress-dependent reduction in photosynthetic
metabolism the factor that inevitably results in reduction of constitutive emissions
and thereby enhanced ROS? These are the questions to which we, armed with
modern molecular, cell, and integrative biology tools, will hopefully soon have a
conclusive answer.
The issue with ozone pollution in current and future atmospheres opens a number
of additional highly relevant issues. Many of the stress-triggered volatile signals are
sensitive to changes in the oxidative nature of the atmosphere through which they
are transmitted (Holopainen et al. 2013). This means that more reactive compounds
can be important for signalling only over shorter distances, while less reactive
compounds can be propagated as ecological signals over longer distances. This may
be the requisite relation given that the atmosphere is oxidative in its fundamental
nature, and compounds with higher reactivity may not be capable of carrying out
their signalling role far from their emission source – especially if the adaptive role is
to elicit top-down controls over herbivory (Trowbridge and Stoy 2013). We are just
now beginning to understand the nature of multitrophic signalling systems (Baldwin
et al. 2006; Dicke et al. 2009; Dicke and Baldwin 2010), and as we gain knowledge
about these systems, which have evolved in atmospheres of fairly constant oxidative
potential, we realize that the atmosphere is changing rapidly due to anthropogenic
influences. Now, the challenge is to determine how these signalling pathways will
change as the oxidative potential of the atmosphere continues to change (Holopainen
et al. 2013).

18.4 What Is Lacking in the Emission Models?

In the atmospheric sciences, simple empirical algorithms dating back to the early
1990s (see the Preface) are often used to predict the source magnitude of tree-
produced BVOCs and their emissions to the atmosphere. However, there have
been major advancements in the understanding of the factors controlling volatile
emissions at timescales from minutes-to-days-to-seasons and at spatial scales
ranging from cellular-to-global. In particular, gene expression studies have provided
important information on the regulation of key controlling enzyme activities
(Sects. 18.1 and 18.2), while novel physiological (CO2 effects on emissions),
physico-chemical mechanisms (controls by volatility) have been discovered and
quantitative algorithms for all of these effects have been developed for inclusion
in large-scale computer models (Sects. 18.2 and 18.3, Grote et al. 2013).
Furthermore, while previous research and models have focused only on constitu-
tive emissions, there has been increased understanding that BVOC emissions can be
triggered by various biotic and abiotic stresses in nearly all previously observed
species (Sect. 18.3, Niinemets et al. 2010b). Quantitative relationships between
the severity of the stress and the rate of emission have been reported for several
stresses (Beauchamp et al. 2005; Loreto et al. 2006; Copolovici et al. 2011, 2012),
518 Ü. Niinemets and R.K. Monson

suggesting that these interactions can be developed in models; although it might still
be difficult to predict precise BVOC elicitation stress thresholds (Niinemets 2010).
Furthermore, there is a scarcity of studies of stress versus induction relations in trees.
For induced emissions, new quantitative models are just beginning to emerge (Grote
et al. 2013 for a construction of a quantitative model for insect herbivory elicited
emissions) opening the frontiers for future components of ecological complexity to
be included in BVOC emissions modelling.
The chapter by Grote et al. (2013) has provided a clear path through the
historical development of the algorithms underlying most existing models of BVOC
emission. Many of these models are well justified in the relation of their components
to fundamental metabolic processes and their interactions with the environment.
However, these models are often isolated as single-factor algorithms. There is room
for further exploration of a mathematical framework within which processes can
interact and within which higher-order feedbacks can be represented. There is also
room for a ‘systems’ approach to modelling and for the exploration of new analytical
expressions that may bring together, in mathematical form, the true synergies that
exist in metabolic form (e.g., Harrison et al. 2013).
Apart from leaf-level improved algorithms, scaling up from leaf to canopy and
landscape has received relatively little attention in BVOC community, reflecting the
difficulties of measuring BVOC emission fluxes from ecosystems due to lack of
suitable fast BVOC sensors (Hewitt 1999). Despite these difficulties, basic canopy
radiative transfer features were described in several early algorithms (Guenther et al.
1994, 1995; Baldocchi and Meyers 1998). With development of methods aimed
at canopy-scale flux measurements of reactive compounds such as relaxed eddy
accumulation (Bowling et al. 1998; Ciccioli et al. 2003), and the invention of ‘fast-
response’ BVOC sensors for use in measuring single compound eddy covariance
(Guenther and Hills 1998) or for multiple compounds sampled interchangeably
(“disjunctly”) (Karl et al. 2002; Grabmer et al. 2004), and with development of
recent technology for true eddy covariance measurement of multiple trace gases
(Müller et al. 2010), ecosystem level trace gas fluxes are well within the realm of
accurate measurements, allowing for the testing of scaled-up leaf models at the
ecosystem and landscape scales (Guenther 2013; Niinemets et al. 2013). While
the canopy parameterization schemes could be successfully validated in many
cases (Guenther 2013 for a review), there are still inherent uncertainties associated
with the temporal resolution of canopy flux data, standardized emission factors
for dominant species, descriptions of canopy structure and key emitting species
coverage such that different leaf-level algorithms, once consistently parameterized,
can not always be conclusively compared (Niinemets et al. 2013). As it becomes
hard to say what is the best model, the selection of a model is often driven by
practical reasoning rather than by objective criteria of model performance. However,
if we are to design models with the capability for projection of emissions into future
climate regimes or in regimes of elevated atmospheric CO2 concentration, we must
design our models to explicitly accommodate the longer-term acclimation controls
over emissions to climate as well as to atmospheric [CO2 ].
18 State-of-the-Art of BVOC Research: What Do We Have and What Have. . . 519

Beyond the canopy and landscape scales, many uncertainties remain with models
at the global scale (Arneth et al. 2008; Ashworth et al. 2013). Major components
of the uncertainty include difficulties with scaled-up emission potential estimates,
landcover types and climatic driver databases that affect emissions in non-linear
fashion, but are often linearized during averaging to the global scale (Ashworth et al.
2013; Guenther 2013). At the global scale, novel methods to test model predictions
by top-down inverse modelling, using for example formaldehyde columns as a
proxy of isoprene formation, have been developed (Palmer et al. 2003, 2006;
Ashworth et al. 2013). However, at this level of resolution, it becomes especially
difficult to sort out the emission algorithm effects on predictions. Instead of showing
the differences among the emission algorithms per se, the model intercomparisons
conducted so far have highlighted major inconsistencies in model parameterization
(Arneth et al. 2011; Ashworth et al. 2013). This certainly underscores the need for
greater care in model parameterization and selection of climatic driver datasets.
Accurate consideration of these fundamental, long-term mechanisms and re-
sponses is essential to quantitatively explore the key feedbacks between global
emissions and global change postulated recently. The global feedbacks involving
BVOCs include BVOC effects on tropospheric ozone level altering the productivity
of non-emitting species, BVOC effects on the formation of secondary organic
aerosols (SOA) and cloud condensation nuclei (CNN) altering the amount of diffuse
radiation and plant productivity and further modifying solar radiation penetration
and the rate of atmospheric [CO2 ] and temperature increase (Kulmala et al. 2004,
2013; Lerdau 2007; Sitch et al. 2007; Mercado et al. 2009; Arneth et al. 2010;
Lathière et al. 2010; Ashworth et al. 2013). These global feedbacks surely constitute
currently an area of high uncertainty, and have not been quantitatively tested to our
knowledge. In this book, Kulmala et al. (2013) provide a first quantitative test of
large-scale feedbacks involving BVOC emissions, plant productivity, SOA, clouds,
atmospheric [CO2 ] and temperature in boreal conifer forests that are known to be
responsible for high aerosol loading in the atmosphere (Tunved et al. 2006). The
analysis demonstrates that BVOC emissions can play a major role in large-scale
biosphere-atmosphere interactions and that the postulated feedback loops between
BVOC emissions and atmospheric processes are likely operative (Kulmala et al.
2013). Their chapter not only demonstrates that BVOC models are ultimately
becoming robust enough to be used in Earth system models, but also indicates
that the knowledge in physical science has been reaching forward to be able to use
BVOC emissions to predict SOA and cloud formation. Clearly, there is still a long
and possibly a winding path to true Earth system models capable of simulating large-
scale climate-vegetation processes with high degree of certainty, but we believe that
with this exercise an important milestone has been reached.
One issue yet to be reconciled in global emission models is the heterogeneity
created in vegetation distributions due to urban and suburban development (Hewitt
et al. 2009; Owen et al. 2013). Ecology in the future cannot pretend as though the
globe is covered with natural forests, and ignore the fact that human settlement and
agronomic activities will continue to re-distribute forests through the development
of feedstocks for second-generation biofuels and through horticultural decoration.
520 Ü. Niinemets and R.K. Monson

Areas in which non-emitting species used to occupy vast tracts of land are now being
converted into short-rotation forests to be used in harvesting cellulose for ethanol
production. Most of the species used in these forests emit BVOCs of various types
at relatively high rates (Owen et al. 2013). This trend will continue well into the
current century, and is certain to modify our current projections of global BVOC
emission patterns. It would seem imperative that new databases and inventories be
developed that can inform the BVOC modelling community as to the location and
rates of spread of agroforestry activities.

18.5 Outlook

The sources of volatile organics can be both anthropogenic and biological, but
biological emissions, and in particular, emissions by trees, are more than an order
of magnitude higher than anthropogenic emissions. Furthermore, biogenic, high
molecular mass, compounds and their oxidation products have the potential to
enter the aerosol phase of the atmosphere through condensation on the surfaces of
secondary volatile organic aerosols. This process of aerosol production has profound
implications for the global radiation budget and climate. In the chapters of this book,
control over BVOC emissions has been discussed at numerous scales and within
the context of ecological, evolutionary, atmospheric and climatic perspectives.
A complete understanding of the primary influences and coupled feedbacks between
tree BVOC emissions and future states of the Earth system will require even further
synthesis among BVOC control processes.
The chapters in this book make clear that we have entered a new and broader
realm of conversation and collaboration within the BVOC research community.
Ecologists with interests in multitrophic interactions are interacting with atmo-
spheric chemists interested in the degradation of ecological signalling pathways.
Molecular biologists interested in gene sequences, induction of promoter activity
and modulation of enzyme activity are interacting with physiologists and bio-
chemists interested in the complexities of biochemical pathway controls and the
tradeoffs between enzyme activity and substrate production as factors determining
the shapes of emission responses to environmental variation. All of these communi-
ties are interacting with modelers interested in providing greater biological reality to
their emission projections, at the local, regional and global scales. The emergence
of new tools such as those used for transgenic manipulation, and the detection of
novel chemical species, have opened up new opportunities to validate models for
control over BVOC emissions at the scales of chloroplasts, leaves, canopies and
even the entire globe. In all of these interactions, we see evidence of new intellectual
energy as the community of scientists considering BVOC emissions continues to
grow, in both the entrainment of established researchers and the recruitment of a
new generation of researchers. In ending this volume, the editors cannot help but
think that this surely must be one of the most intellectually stimulating and broad
academic disciplines with which to be affiliated!
18 State-of-the-Art of BVOC Research: What Do We Have and What Have. . . 521

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Editors Biography

Professor Ülo Niinemets did his Ph.D. in plant ecophysiology at the University
of Tartu, Estonia and conducted postdoctoral work at the University of Bayreuth,
Germany; University of Antwerp, Belgium and Centro di Ecologia Alpina, Trento,
Italy. His past international faculty appointments include Erskine fellow (2002,
Canterbury University, New Zealand), annual G. P. Wilder Chair (2006–2007,
University of Hawaii, USA) and F. C. Donders Chair (Utrecht University, The
Netherlands). He is currently the head of the Department of Plant Physiology
at the Estonian University of Life Sciences where he is in charge of a research
team dealing with quantification and predictive modelling of plant carbon gain
and trace gas exchange from leaf to ecosystem, landscape and biome scales under
globally changing climates. He has collaborated with more than 450 scientists
from 40 countries and has co-authored more than 200 international articles and
book chapters. He serves the community as an editor or editorial board member
of multiple international scientific journals and as a steering committee member of
several international programs.
Professor Russell K. Monson is Louise Marshall Foucar Professor in the School of
Natural Resources and the Environment and the Laboratory for Tree Ring Research
at the University of Arizona. He is Professor Emeritus in the Department of Ecology
and Evolutionary Biology at the University of Colorado, and Affiliate Scientist in the
Atmospheric Chemistry Division at the National Center for Atmospheric Chemistry,
Boulder. He has published over 175 articles and book chapters on topics including
photosynthetic metabolism in plants, the emission of volatile organic compounds
from leaves and forests, and forest carbon and water cycles. He has conducted
research as a Humboldt Fellow, a Guggenheim Fellow and a Senior Fulbright
Fellow, and has served as Editor-in-Chief or editorial board member for several
international research journals.

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 529
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8,
© Springer ScienceCBusiness Media Dordrecht 2013
Index

A Acetyl coenzyme A (Acetyl-CoA), 50, 98, 125,

ABA. See Abscisic acid (ABA) 157, 168–170
cis-Abienol synthase, 70 Acetylene, 439
Abies Acetylphosphinate, 168
A. alba, 322 cis-Acting element, 80
A. balsamea, 70 Activation energy, 133
A. concolor, 79 Active center, 80
A. grandis, 54–60, 63–65, 67, 69–71, 75, Acute exposure, 269
79 Acyclic monoterpene, 71, 72, 123, 130,
Abietadiene synthase, 60, 63, 65, 67, 68, 75 510
Abiotic stress, vii, 24, 48, 72, 77–79, 108, Adaptation, 38, 77, 80, 81, 154, 160, 237, 238,
121, 124, 154, 170, 171, 209–227, 246, 244, 249, 265, 269, 292, 308, 383, 462,
316, 339–340, 423–424, 433, 510, 511, 475
515–517 Adenosine triphosphate (ATP), 51, 123, 128,
Abiotic stressor, 3, 9 133–135, 137, 157, 164, 223, 238, 264,
Above-canopy environment, 367, 392, 265, 270, 326–328, 331, 362, 457, 458,
398–401 512, 513
Abscisic acid (ABA), 98, 101, 124, 184–187, Adenostoma fasciculatum, 260, 266
205, 243 ADH. See Alcohol dehydrogenase (ADH)
Acacia Advanced very high resolution radiometer
A. nigrescens, 259, 266, 425 (AVHRR), 402
A. nilotica, 425 Adventitious roots, 242, 244
Acclimation, vii, 156, 213–216, 226, 238, 242, Aerenchyma, 242, 244, 247–249, 516
246, 248, 255, 256, 263–266, 270, 316, Aerial photographs, 430, 431
317, 319, 325, 331–336, 358, 379, 456, Aerosols, vi, 12, 49, 171, 211, 224, 296, 301,
500, 518 305, 392, 399, 416, 453, 456, 463, 465,
Acclimation mechanisms, 256, 317 469–471, 489–504, 519, 520
Accuracy, 161, 383, 392, 405–407, 410 model, 470, 471, 496, 501
Acetaldehyde, 121, 154, 168–170, 172, 190, optical depth, 463
191, 210, 214, 218, 239, 240, 242–245, particle number concentration, 493
248, 249, 422, 516 phase, 453, 470, 496, 520
Acetaldehyde emission, 168–170, 237–245 Agroforest, 96, 171, 342, 416, 417, 420, 424,
Acetic acid, 120, 186, 187, 190, 191, 239, 240, 427, 432, 435, 520
245, 248, 422 Agroforestry plantation, 415–441
Acetone, 191, 211, 213, 218, 422, 472 Aircraft measurements, 406, 431
Acetyl-CoA. See Acetyl coenzyme A Air/lipid-phase partition coefficient, 203

Ü. Niinemets and R.K. Monson (eds.), Biology, Controls and Models of Tree Volatile 531
Organic Compound Emissions, Tree Physiology 5, DOI 10.1007/978-94-007-6606-8,
© Springer ScienceCBusiness Media Dordrecht 2013
532 Index

Air pollution, vii, 13, 210, 219, 253–276, Asymmetric function, 332, 360
286–288, 294, 298, 299, 304–306, 320, Atmospheric chemistry, vi, 14, 227, 272, 318,
358, 405 344, 409, 416, 417, 441, 453, 463, 470,
modelling, 253–276 473, 476, 490, 504, 527
Air quality, 49, 96, 272, 286, 391, 392, 410, Atmospheric CO2 concentration, 170, 171,
416, 420, 440, 454, 466, 473, 490 210, 225, 258, 263, 266, 273, 302, 329,
Air quality model, 205, 273, 391, 405, 454 367, 468, 489–504, 510, 518
Alarm pheromone, 33, 304 Atmospheric composition, viii, 255, 257, 275,
Albedo, 171, 224, 490, 502, 503 453, 464, 465, 470, 473
Alcohol dehydrogenase (ADH), 168 Atmospheric lifetime, 210, 295, 298, 301, 452,
Alcoholic fermentation, 238–242, 245 453, 469
Aldehyde dehydrogenase (ALDH), 168–170, Atmospheric pollutant, 2, 286, 297, 303, 307
240 Atmospheric quality, 305
Aleppo pine, 217, 269 ATP. See Adenosine triphosphate (ATP)
Alicyclobacillus acidocaldarius, 65, 67 Attractant, 28, 29, 32, 36, 79, 292
Alien species, 419
Allelopathy, 22
Allosteric modification, 154, 162 B
4-Allyl anisole, 293 Baccharopelma dracunculifoliae, 293
Alnus glutinosa, 33, 79, 223, 239, 242, 246, Bacillus thuringiensis, 302
288, 297, 343, 344 Bacterial artificial chromosome, 54
Alternative carbon source, 216 Ball-Berry equation, 367
Amazon, 248, 397, 453, 474 ’-Barrel, 63
Amazonian, 239, 242, 245, 247, 256 Basal emission, 104, 360, 422
Amino acid sequence, 65, 66, 68, 129 Basal emission rate (BER), 155, 379, 455, 459,
ANNs. See Artificial neural networks (ANNs) 461, 462
Anoxia, 168, 237–239, 241, 244, 516 Beer’s law, 395
Anthropogenic emission, 406, 464, 490, 500, Belowground organisms, 27
501, 503, 504, 520 Benzene, 436, 439
Antibiotic function, 24 Benzenoid, 22
Anti-Markovnikov addition, 72 Benzyl cyanide, 32
Antioxidant, 48, 109, 221–223, 226, 271 BER. See Basal emission rate (BER)
Antirrhinum majus, 71 Betula, 291, 306, 340
Antixenotic function, 24 B. nana, 106
Aphid, 33, 34, 36, 287, 288, 302, 304 B. pendula, 61, 106, 107, 214, 260, 266,
Apoptosis, 247 271, 288, 297, 330
Apple, 34, 110 B. pubescens ssp. Czerepanovii, 294, 297
Aqueous phase. See Liquid phase Bifunctional diterpene synthase, 69, 70
Aqueous phase conductance. See Liquid phase Bifunctional synthase, 67
conductance Big-leaf model, 394, 438
Arabidopsis, 53, 61, 69, 101, 102, 104–108, Biochemical diversity, 418
127, 128, 131, 132, 137, 139, 142, 159, Biodiversity, 274, 417–419
238 Bio-engineering, 255
Aradus cinnamomeus, 287 Biofuel, 13, 49, 108, 171, 417, 441, 519
Aristolochene synthase, 65 Biogenic emission inventory, vi, 455
Arms race, 22, 292 Biogenic Emission Inventory System (BEIS),
Artemisia vi, 397, 401, 405, 407, 455
A. annua, 99 Biogenic Emission Inventory System2
A. tridentata, 291 (BEIS2), 405, 407
Artificial neural networks (ANNs), 61, 456, Biogenic Emission Inventory System3
457 (BEIS3), 406
Arundo donax, 263 Biogenic volatile organic compounds (BVOCs)
Asobara tabida, 299 emission, vii, 6, 24, 27, 28, 31, 108, 142,
Associative resistance, 291 155, 156, 182, 186, 193, 199, 212, 213,
Index 533

215, 218, 224, 225, 248, 255–259, 267, scale, 358, 359, 383, 392–395, 398, 406,
268, 271, 274, 276, 289, 303, 318–320, 410, 423, 424, 427, 428, 435–436,
326, 330, 339, 340, 345, 383, 391–395, 439–441, 518
398–401, 404–410, 418–438, 440, 441, structure, 336, 394, 395, 518
453, 456, 462, 464, 469, 471–473, 475, Carbocation, 63, 68, 72, 131
476, 491, 496, 497, 501–503, 514, 518 Carbon cost of isoprenoid emission, 327
induction, 341 Carbon di oxide (CO2 )
Biological control, vi, vii, 120 compensation point, 136, 322–323, 361
Biome, viii, 5, 6, 460, 462 inhibition, 137, 273, 465
Biosynthesis rate, 326, 516 response, 136, 164, 216, 262–265, 326–329
Biotic stress, vi, vii, 79–81, 109, 120, 219, 227, sensitivity, 326, 513
340, 424, 512, 515 3-Carene synthase, 80
Biotic stressor, 2, 31, 290 Carica papaya, 53, 77, 96
’-Bisabolene synthase, 58, 64, 65, 67, 69, 71 Carotenoid, 48, 51, 98, 100–103, 124, 134,
Black poplar. See Populus, P. nigra 159, 221, 223
Bloch-Lynen pathway, 157 “-Caryophyllene, 59, 105, 295, 298
Blue haze, v Catalytic site, 63, 65
Boreal, 5, 13, 29, 96, 410, 468, 469, 491, CCN. See Cloud condensation nuclei (CCN)
493–496, 499, 503, 519 Cd, 302
Borneol, 289 Ceanothus greggii, 260, 266
Bornyl di phosphate synthase, 64, 67 Chaitophorus stevensis, 304
Bottom-up approach, 318, 428–429, 432 Chamaecyparis obtusa, 423
Bottom-up defence, 24, 29 Chemical cue, 299
Boundary layer, 182, 271, 395, 428, 470 Chemical defence, 23, 33, 78
Bradyrhizobium japonicum, 73 Chemical diversity, 50, 80
Brassica Chemical signalling, 22, 286
B. napus, 302 Chemistry-climate model (CCM), 470
B. nigra, 36 Chemosensory sensilla, 37
B. oleracea, 302 Chemotype, 27, 189
B. oleracea ssp. capitata, 302 Cherry, 110
Brassinosteroid, 98 Chewing insects, 31, 288
Breakage of storage structures, 338 Chiral isomers, 124
Bruchins, 30, 32 Chloroplastic signal peptide, 54
Bryophyta, 9 Chloroplasts, 51, 63, 69, 70, 99, 101–104,
Bulk canopy approach, 393–394, 396 120, 125, 126, 128, 130–132, 137–138,
Bursts of methanol emission, 186, 188–189, 141, 157–160, 162–164, 169, 223, 264,
198, 201 318, 321, 324, 327–329, 331, 457, 458,
BVOCEM, 460 512–514, 520
BVOCs. See Biogenic volatile organic Chronic O3 exposure, 270
compounds (BVOCs) Chrysonotomyia ruforum, 34
1,8-Cineole, 110, 187, 188, 190, 191, 289
Circadian change, 139
C Circadian clock, 104
Cabbage, 302 Circadian control, 7, 159, 514
Cabera pusaria, 79, 343, 344 Circadian rhythm, 102, 159
Camphene, 55, 57, 191 Cistus incanus, 322
CANOAK, 396 Citrus
Canopy C. clementina, 76, 77
effects, 256 C. limon, 56, 58, 110, 247
emission flux, 394 C. paradisi, 34
environment, 392–401, 409, 410, 441 C. sinensis, 110
13
location, 141 C-labelling, 103, 157, 163, 165, 166, 168,
model, 367, 368, 371, 379, 383, 394–398, 203, 204, 264, 512
400, 410, 427, 438, 455 Classification, 67, 72, 155, 430–433, 440
534 Index

Class I terpenoid synthase, 63, 68 Cropping, 420–422

Class II terpenoid synthase, 63 Crop plantation, 416
CLAW hypothesis, 490–493 Cross-resistance, 31
Climate Cross-talk, 30, 36, 50, 98, 103, 154, 158–160,
change, 11–14, 24, 120, 255, 286, 429, 441, 172, 341
461, 467–468, 490, 492, 500 Cryptomeria japonica, 54, 423
feedback, 489–503 Crystal structure, 67, 68, 72
forcing, 468, 493 Cu, 302
Cloud Cunninghamia, 440
albedo, 224, 490, 502, 503 Cupressus forbesii, 426
cover, 399, 470 Cuscuta pentagona, 291
droplet number, 466, 491, 502 Cu stress, 302
formation, 471, 489, 519 Cuticular resistance, 182
lifetime, 465 Cutting, 136, 422
Cloud condensation nuclei (CCN), vi, 49, 224, Cyclosativene, 59
465, 470, 490, 491, 493, 496, 497, 4-(Cytidine 50 -diphospho)-2-C-methyl-
500–503 D -erythritol (CDPME), 102, 126
Cloudiness, 395, 396, 402, 469–470, 495 4-(Cytidine 50 -diphospho)-2-C-methyl-
CO2 . See Carbon di oxide (CO2 ) D -erythritol kinase (CMK), 102,
13
CO2 -labelling. See13 C-labelling 127
Coagulation sink, 497 Cytochrome P452-dependent, 51
CO column, 463
Codon usage, 54, 63
Coevolution, 38–39, 292–293 D
Coevolutionary process, 28, 39 Dactylis glomerata, 170
Combined stress, 211, 219, 307 Dark respiration, 261, 264
Community composition, 38, 171, 274 DDXXD motif, 63, 64
Compartmentalization, 159 Deception, 33
Condensable vapors (CS), 497, 498 Deep sequencing, 527
Condensation sink (CS), 491, 494–496, 498, Defence, 2, 3, 8, 22–27, 29–37, 39, 48, 78, 79,
499 211, 222, 225, 226, 287, 290–293, 296,
Conifers, 2, 9, 27, 34, 48, 54, 70, 78, 79, 107, 302, 305, 316, 338, 340, 515
166, 181, 186, 203, 204, 266, 288, 290, Defoliating insects, 287
293, 305, 318, 321, 338, 341, 371, 380, Defoliator, 37, 287, 293, 299
402, 408, 459, 491, 492, 519 Demethylation, 167, 172, 247
Conium maculatum, 293 Dendroctonus, 25
Constitutive defence, 22 Dendroctonus ponderosae, 403
Constitutive emission, 316, 337–341 1-Deoxy-D -xylulose 5-phosphate (DOXP),
Constitutive emitter, 339 50–51, 68, 96, 99–101, 157, 158, 264,
Convergent evolution, 72 318, 375, 512
ent-Copalyl di phosphate, 73 1-Deoxy-D -xylulose 5-phosphate (DOXP)
Copalyl di phosphate (CDP), 64, 69, 70 synthase, 101, 161
Copalyldiphosphate synthase, 73 1-Deoxy-D -xylulose 5-phosphate
ent-Copalyl di phosphate synthase, 69, 73 reductoisomerase (DXR), 100–
Correlation, 104, 160–163, 186, 187, 189, 204, 102, 108, 126–128, 134
217, 341–243, 248, 263, 264, 331, 342, Deoxyxylulose (DOX), 100, 101, 161, 162
359, 362–364, 372, 379, 400, 498, 500, Detoxification trait, 292
512, 516 Developmental effects, 104, 108, 140, 172,
Cotesia 210
C. melitaearum, 304 Diachasmimorpha longicaudata, 34
C. vestalis, 300, 302 Diffuse light, 12, 401, 471
C-ratio model, 327, 361, 363–366, 368, 370, Diffuse PPFD, 400, 401, 471
375–379, 383 Diffuse solar radiation, 224, 465, 469–471,
Cretaceous, 10, 266 493, 495, 503
Index 535

Diffusion Earth system models, 391, 392, 403, 404,

constant, 194 519
gradient, 338, 514 ECHAM5-HAM, 495
path, 194, 197 Ecosystem engineering, 224
pathway length, 195 Ecosystem-level emission, 380, 455
resistance, 167, 213, 215, 321 Eddy covariance, 368, 380, 383, 397, 406, 407,
Diffusivity, 184, 196, 366 410, 435, 462, 495, 498, 518
Digital terrain model, 432 Egg deposition, 30–32
Dimethylallyl di phosphate (DMADP), 8, Elaeis guineensis, 13, 96, 104, 159, 171, 419,
50, 51, 55, 68–71, 97–102, 105, 121, 425, 435, 439, 440, 461
123, 125, 126, 128, 130, 133–137, 141, Electro-antennogram (EAG), 297
155–158, 160–164, 172, 218, 221, 223, Electron transport, 137, 220, 270, 324–326,
263–266, 270, 326–329, 458, 512–514 329, 362, 378, 454, 458, 474
4,8-Dimethylnona-1,3,7-triene (DMNT), 214, Elevated [CO2 ], vii, 141, 225, 255–258,
288, 295, 297, 340 260–267, 271–274, 302–304, 467, 492,
Dinitrogen pentoxide (N2 O5 ), 307 493, 500
Diprion pini, 34 Elevated [O3 ], 105, 261, 267–271, 275, 300,
Direct defence, 3, 29, 48, 78, 79 302–304
Disulfiram, 168, 169 Emission
Diterpenes, 10, 48, 49, 51, 52, 54, 60, 61, 63, algorithm, 319, 371, 394, 398, 406, 408,
65, 67, 69–73, 75, 78, 79, 99, 100, 107, 456, 459, 461, 519
128, 159, 301 capacity, vi, 5, 9–11, 104, 120, 136,
Diterpene synthase, 10, 69–73, 79, 128 139–141, 156, 167, 210, 257, 265, 266,
Diversification, 1–14, 510, 511 321, 325, 331–334, 337, 366, 383,
DMADP. See Dimethylallyl di phosphate 399–400, 492
(DMADP) diversity, vii, 475, 509–512
DMADP pool size, 263, 265, 266, 328, 330, factor (see Emission, potential)
331, 512, 513 model, vii, viii, 4, 155, 186, 193, 205,
DNA sequence, 5 206, 316–321, 326, 330, 331, 335, 337,
“-Domain, 64–70, 73, 75, 76 338, 358–372, 374, 378, 380, 383, 392,
’-“-”-Domain synthase, 73 393, 395, 398–401, 404–408, 410, 428,
’-Domain synthase, 65 454–460, 462–464, 469, 470, 472, 473,
“-”-Domain synthase, 65 475, 517–520
Domestication, 9 potential, 5, 155, 258, 320, 332, 334–336,
Double-exponential model, 192 341, 360, 369, 371, 373, 378, 381, 382,
Downward solar radiation, 399, 400 393, 396, 397, 403, 406, 408–410, 420,
Drosophila subobscura, 299 423, 424, 428, 429, 433, 437, 438, 457,
Drought, vii, 12–14, 30, 105, 156, 162, 163, 460, 474, 518, 519
171, 187, 210–213, 215–219, 225, 226, rate, 5, 24, 48, 123, 155, 182, 213, 237,
254, 260–263, 265, 316, 317, 319, 330, 257, 289, 317, 358, 407, 418, 454, 510
337–339, 358, 369, 375, 399, 409, 421, source, 300, 307, 371, 428, 517
424, 428, 464, 465, 471, 510, 516 Empirical model, 120, 134, 340, 358, 454–456,
Duplicated genes, 77 461
Duration of flooding, 244–245, 516 Enclosure measurements, 371, 410, 494
(N/D)DXX(S/T)XXXE motif, 63, 64 Endoparasitoid, 294
Dynamic emission model, 360, 366–367, 377 Energy budget, 395
Dynamic enclosure systems, 434 Enthalpy
Dynamic model, 188–205, 359–361, 370–371, of activation, 323
373, 375–378, 383, 514 of de-activation, 323
of volatilization, 366
Entropy, 323
E Epirrita autumnata, 294
Earth system, v, vi, viii, 39, 410, 452, 453, Episyrphus balteatus, 304
464–472, 520 Escherichia coli (E. coli), 54–63, 127
536 Index

Ethanol Ferredoxin, 51, 123, 128, 135, 143

concentration, 240, 241, 244 Fertilizer, 420–422, 424
emission, 168, 239, 242, 245 Fick’s first law, 182, 184
Ethylene, 31, 210, 245, 247, 249, 341 Field experiments, 257–258, 270, 515
Ethylene emission, 245 Fitness, 9, 22, 25, 26, 35, 38, 39, 72, 96, 109,
Eucalyptus 154, 212, 217, 227, 286, 291, 292, 511,
E. globulus, 101, 104, 259, 263, 368, 425, 515
426 Flooding
E. grandis, 53, 75, 77 duration, 244–245, 516
E. saligna, 422, 425 tolerance, 223, 238, 239, 241–242, 247
Eucalyptus spp., 13, 96, 110, 171, 255, 440 Floral emission, 297
Evaporative cooling, 337 Fluidity, 48, 220
Evapotranspiration, 12, 464, 465, 472 Flux measurements, 359, 370, 378, 380, 383,
Evergreen, 6, 13, 14, 49, 70, 78, 218, 319, 394, 397, 406, 407, 435, 439–441, 475,
320, 326, 333, 336, 341, 358, 359, 365, 476, 518
367–371, 374, 378, 492 Foliar biomass, 408, 410, 440
Evolution, 3, 31, 104, 130, 225, 292, 453, 503, Foliar density, 28, 409
509 Foraging dynamics, 27
Evolutionary adaptation, 38, 80, 154 Foraging pattern, 36
Evolutionary distance, 68 Forest area, 417
Evolutionary history, 4, 5, 9 Formaldehyde (HCHO), 190, 191, 218, 320,
Excess light, 222 406, 422, 456, 463, 464, 519
Exotics, 419, 420 Formaldehyde (HCHO) column, 463, 519
Exotic species, 417, 419–420, 429, 437 Formic acid, 190, 191
Explained variance, 368, 372, 374, 377–379 Fosmidomycin (FSM), 100, 101, 125–126,
Exponential decay, 192 159, 220, 510
Expressed sequence tag (EST), 54 Fragaria ananassa, 71
Expression pattern, 78 Fragmented vegetation, 437
Extinction coefficient, 395 Fraxinus excelsior, 240
Extreme weather event, 171 Free-air CO2 enrichment (FACE), 257, 259,
261, 262, 268, 303, 493
Frost, 340
F FSM. See Fosmidomycin (FSM)
FACE. See Free-air CO2 enrichment (FACE) Full-length cDNA mining, 54
Fagus sylvatica, 242, 245, 320, 358, 459, 492 Fumigation system, 257
(E)-“-Farnesene, 33, 34, 36, 59, 75, 289, 295, Functional characterization, 49, 53, 54, 61, 71,
304 77
Farnesene synthase, 59, 71 Functional domain, 53, 63–68
Farnesyl di phosphate (FDP), 51, 58, 59, 61, Fungal pathogen, 288, 292, 340
70, 71, 99, 102, 107, 123, 131 Fungal symbiont, 288
Fast isoprene sensor, 142, 435 Fusarium sporotrichioides, 65
FDP. See Farnesyl di phosphate (FDP) Future climate, 274, 429, 468, 473, 475
Feedback(s)
control, 162, 172
cycles, 464, 465, 471, 472, 501 G
loop, 101, 255, 274, 328, 471, 490, 491, Gaia hypothesis, 490
493–504, 519 GAP. See Glyceraldehyde 3-phosphate (GAP)
mechanism, 97, 490, 493 Garcinia macrophylla, 247
Feeding behavior, 343 Gardening, 6
Feeding guilds, 31 GASFLUX, 367, 368
Feeding-promoting parasitoids, 294 Gas/liquid phase partition coefficient, 191,
Felling, 420–422 337, 367
Fermentation, 168, 238–242, 245, 248, 516 Gas phase, vi, 167, 190, 194–197, 199, 201,
Fermentation products, 239–241 202, 205, 271, 294, 337, 367, 470
Index 537

Gas phase conductance, 194 Gossypium arboreum, 65, 67, 68

Gaussian response, 332 GPP. See Gross primary production (GPP)
GDP. See Geranyl di phosphate (GDP) Gradient flux measurements, 397
Gene duplication, 73, 75, 80 Gradient profile, 462
Gene expression, vii, 24, 31, 53, 54, 77, 78, Grand fir, 64, 68
101, 108, 132, 139, 141–142, 153–172, Grapefruit, 34
238, 270, 316, 340, 341, 343, 345, 517 Grassland, 13, 171, 381, 400, 405, 422
Gene-expression response, 155, 156 Green fluorescence protein, 104
Gene family, 55, 56, 58, 60, 66, 75, 77, 80 Green leaf volatiles (GLVs), 25, 31, 168–170,
General circulation model (GCM), 468 172, 210, 214, 215, 218, 222, 224, 226,
Generalist parasitoid, 38 248, 267, 288, 295, 340, 515
Generalists, 28, 38, 291–293 Grey poplar, 67, 68, 104, 107, 109, 141, 218
Genetically modified (GM) plant, 53, 95, 96, Gross primary production (GPP), 273, 490,
104, 105, 108–110, 219, 222, 226–227, 491, 495–499
510, 511 Ground survey, 432–433, 437–438
Genetic diversity, 81 Growth light, 156
Genetic engineering, vi, vii, 49, 95–110
Genome
mining, 53, 75 H
sequence, 80 Habitat fragmentation, 304
Genotypic diversity, 9 Half-life. See Half-time
Geographical isolation, 5 Half-time, 135, 139, 195–198, 204
Geographical variation, 5 Halisdota ingens, 79
Geometric isomers, 124 Harmonia axyridis, 304
Geraniol, 123 HCHO. See Formaldehyde (HCHO)
Geranyl di phosphate (GDP), 51, 55–59, 61, Heat stress, 134, 212–215, 220–222, 224, 226
68–72, 99, 102, 107, 125, 130 Heat transfer conductance, 396
Geranylgeranyl di phosphate (GGDP), 51, 60, Heavy metal pollution, 285, 287, 301–302
61, 70, 71, 73, 99 Heliothis virescens, 36
Germacrene D synthase, 59 Hemiboreal, 359, 371–372, 380, 381
GGDP. See Geranylgeranyl di phosphate Hemiterpenes, 49, 52, 55, 65, 73, 75, 79, 99,
(GGDP) 121–123, 126, 159
Gibberellin, 73, 98, 126 Hemiterpene synthase, 52, 73
Gingko biloba, 70, 261 Henry’s law constant, 166, 190, 191, 193,
Global change, 29, 39, 171, 254, 286, 454, 197–199, 204, 205, 212, 213, 217, 239,
471, 519 240, 243, 337, 367
Global dimming, 471 Herbivore
Global feedbacks, viii, 464, 519 attack, 24, 30, 79, 301
Global emission, 398, 407–410, 460, 461, 463 enemies, viii, 48, 292
Global model(ing), 272, 392, 398, 406, 462, performance, 24, 301
464–473, 475, 476, 496, 497, 503 pressure, 29
Global solar radiation, 395, 396, 399 Heterologous expression, 52–62
Global warming, vi, 273 Hevea
Glucosinolates, 293 H. brasiliensis, 13, 425, 426, 440
GLVs. See Green leaf volatiles (GLVs) H. spruceana, 247
Glyceraldehyde 3-phosphate (GAP), 51, 100, (E)-2-Hexenal, 215, 222
101, 114, 120, 125, 136, 138, 160, 221, cis-3-Hexenyl acetate, 267, 290, 295
264, 325, 331, 458 High temperature, 3, 11–13, 26, 134, 137, 139,
Glycine max, 142 171, 212–214, 223, 262, 273, 364, 399,
Glycolysis, 101, 135, 160, 168, 169, 172, 238, 510–511
264, 516 Himachalene, 59
Glyoxal, 463 HMBDP. See 4-Hydroxy-3-methylbut-2-
GM plant. See Genetically modified (GM) enyldiphosphate (HMBDP)
plant Homeostasis, 159–164
538 Index

Homogalacturonic acid (HGA), 167 Intercellular air space conductance, 183, 184,
Homogeneity, 53, 72, 75, 416 190, 194, 336, 514
Homoterpene, 36, 79, 214, 288, 295, 515 Interferometric radar, 432
Host quality, 26, 511 Interior spruce, 54
Host selection, 24–26, 292 Interspecific signalling, 291
”-Humulene synthase, 59, 64, 71 Intraspecific signalling, 290–291
Humulus lupulus, 71 Invasive, 28, 419
Hybrid aspen, 266, 297 Inverse modelling, 320, 323, 370, 378, 379,
Hybrid poplar, 25, 35, 36, 95, 109, 135, 243, 428, 441, 519
260–261, 297 Ips, 25
4-Hydroxy-3-methylbut-2-enyl di phosphate Irrigation, 420, 421
(HMBDP), 51, 100, 102, 126, 128 Iso-butene, 439
reductase, 51, 126 Isopentenyl di phosphate (IDP), 8, 50, 51, 69,
synthase, 51, 126, 137 97–100, 102, 103, 121, 123, 125, 126,
3-Hydroxy-3-methylglutaryl-CoA (HMG- 128, 130, 157–159
CoA), 50, 98–99, 157 Isopentenyl di phosphate isomerase (IDI), 99,
3-Hydroxy-3-methylglutaryl-CoA (HMG- 100, 102, 126, 128
CoA) pathway, 98, 157 Isopimaradiene synthase, 60
10-Hydroxypinonaldehyde, 295 Isoprene emission, 2, 69, 96, 120, 155, 184,
10-Hydroxypinonic acid, 295 214, 247, 259, 318, 368, 396, 422, 492,
Hygrophyte, 10 508
Hylobius abietis, 27, 28, 289 Isoprene fumigation, 510
Hyperparasitoid, 293 Isoprene synthase, vii, 10, 55, 61, 65, 67–71,
Hypersensitive response, 211, 222 78, 100, 102–106, 126–134, 139–142,
Hyperspectral imaging, 431 155, 158–164, 213, 214, 217, 218, 223,
Hyphantria cunea, 33 226, 265, 266, 270, 323, 329, 330, 332,
Hyposoter horticola, 304, 305 458, 474, 510, 512–514
Hypostomatous, 184 Isoprene synthase promoter, 78, 514
Hypoxia, 237, 238, 244 Isoprene synthesis, 104, 126, 133, 136–138,
218, 272, 326, 329, 474
Isoprenoid synthesis pathways, 264, 458,
I 474
IDI. See Isopentenyl di phosphate isomerase
(IDI)
IDP. See Isopentenyl di phosphate (IDP) J
Igapó, 247 Jasmonic acid (JA), 31, 32, 166, 290, 302
Indirect defence, 25, 30, 33, 35–37, 48, 78, 340 Juglans
Induced defence, 3, 22, 25, 29, 515 J. californica, 342
Induced emission, 3, 37, 210, 212, 267, 289, J. regia, 342
302, 316, 317, 339–345, 515–518 Jungermannia subulata, 73
Induced resistance, 3, 7, 22
Induced response, 22, 26, 27, 29, 32, 35, 36,
248
Infochemical, 22, 25, 37, 511 K
Insect Kaurene, 73, 75, 126, 129
learning, 24 ent-Kaurene, 73, 75
outbreak, 285, 287 ent-Kaurene synthase, 73
perception, 3, 4, 28, 37–39 Kinetic constant, 126, 193, 195, 366
Insect-inducible promoter, 80 Kinetic constraint, 155, 162, 163
Instantaneous emission, 155, 321, 331 Km , 61, 68, 70, 128, 130, 141, 513
Interactive effects, 12, 303, 330 Koinobiont, 293
Intercellular air space, 183, 184, 190, 194, 247, Krebs cycle, 516
325, 337, 514 Kudzu, 61, 142, 160, 423
Index 539

L Light dependence(y), 134, 167, 182, 186, 297,

Laboratory experiments, 257, 259, 264, 267, 318, 321–326, 358, 361, 368, 370, 371,
475 393, 408, 427, 454, 459, 512, 513
Lactic acid, 238 Light-dependent monoterpene emission, 192,
Lactic acid fermentation, 238 220, 225, 327, 511
Laetia corymbulosa, 239 Light Distance And Ranging (LiDAR), 429,
LAI. See Leaf area index (LAI) 430
Lamiaceae, 459 Light-limitation, 408
Landcover, 392, 400–405, 409, 416–417, 419, Limonene, 9, 55–58, 67, 71, 75, 123, 129,
430, 453, 455, 474, 519 131, 187–191, 197, 260, 267, 269, 289,
Landcover types, 381, 403, 405, 431 295–297
Landsat, 431, 432 Limonene synthase, 55, 56
Landscape, viii, 4, 171, 224, 357–383, Linalool, 56, 58, 59, 71, 75, 79, 102, 123, 129,
391–410, 416, 427, 431–433, 502, 518, 130, 187, 188, 190, 191, 203, 204, 289,
519 295, 297, 340
Landscape BVOC emission, 170–171 Linalool/nerolidol synthase, 70, 71, 106
Landscape-scale flux measurements, 435 Linalool synthase, 56, 70, 71, 79, 106
Land surface model, 402 Lipid peroxidation, 222, 224, 269
Land-use change, 13, 171, 272, 415, 416, 429, Lipid phase, 189, 190, 192–196, 201, 203, 204,
440, 441, 453, 490 292, 360, 366, 367, 515
Larix sibirica, 426 conductance, 189, 191, 192, 194, 195, 203
Last glacial maximum, 263, 273, 466, 468, 469 pool, 196, 204, 366
Latent heat, 171, 257, 272, 395, 396, 416 Lipid solubility, 3, 193
Lavandula latifolia, 101 Lipoxygenase (LOX), 169, 210, 246, 249, 267,
Layered canopy, 367, 428 268, 515
Layered canopy model, 379 Lipoxygenase pathway, 246, 267, 515
Leaf Liquidambar styraciflua, 186, 259, 261, 326,
age, 139, 331–334, 336, 423, 427, 433, 458
455, 461 Liquid phase, 165–167, 190–192, 194–198,
area profile, 257 200, 201, 203, 213, 222, 240, 243, 301,
boundary layer, 182, 183 337, 366, 367, 514, 520
clumping, 394, 396 conductance, 197, 198, 337
development, 104, 108, 140, 172, 332, 336, pool, 200, 218, 366
367 LMA. See Leaf dry mass per unit area (LMA)
energy balance, 394–396 Loblolly pine, 34, 54, 201, 202
expansion, 29, 167, 247, 336 Localized fumigation (LOF) system, 257, 268,
orientation, 394 269
temperature, 26, 132, 133, 161–163, 182, Local response, 35, 80, 399, 454
183, 185, 187, 188, 200, 202, 214, 265, Lodgepole pine, 79
272, 291, 321, 322, 326, 335, 340, 360, Logging residue, 422
362, 364–367, 392, 395, 396, 398, 433, Longiborneol, 59
434, 454 Longicyclene, 59
Leaf area index (LAI), 263, 272, 380, 394, 395, Longifolene synthase, 59
397, 401–404, 406, 408–410, 433, 437, Longipinene, 59
438, 492, 493 Long-term control, 155–170
Leaf dry mass per unit area (LMA), 433 Longwave radiation, 395
Learned behavior, 38 Lovastatin, 99, 103
Learning behavior, 38 LOX. See Lipoxygenase (LOX)
Levopimaradiene, 60, 75 LOX products, 246, 268, 269
Levopimaradiene synthase, 60, 70 LPJ-GUESS, 460, 461
LiDAR. See Light Distance And Ranging Lupinus arboreus, 293
(LiDAR) Lycopersicon esculentum, 101
Light-dark transition, 168, 169 Lymantria dispar, 33, 290, 302
540 Index

M 2-Methyl-3-buten-2-ol (MBO), 51, 55, 75, 121,

Magnolia, 70 129, 130, 132
Magnoliophyta, 5, 9, 10 2-Methyl-3-buten-2-ol synthase, 55
Malacosoma disstria, 36, 299 Methyl chavicol, 210
Malus domestica, 53, 59, 71, 110 2-C-Methyl-D -erythritol 2,4-cyclodiphosphate,
Manduca sexta, 107 102
Mangifera indica, 27–28, 34 2-C-Methyl-D -erythritol 2,4-cyclodiphosphate
Mango. See Mangifer indica synthase (MDS), 100, 102, 127
Mapping, 416, 430–432 2-C-Methyl-D -erythritol 2,4-cyclodiphosphate
Markovnikov addition, 72 (MEcDP) synthase, 126, 128, 135
Massively parallel signature sequencing, 53 2-C-Methyl-D -erythritol 4-phosphate (MEP),
Maximum carboxylase activity of Rubisco, 125
329 2-C-Methyl-D -erythritol 4-phosphate
Mean absolute error (MAE), 372–374, 378, cytidylyltransferase, 127
379, 382 2-C-Methyl-D -erythritol 4-phosphate/1-deoxy-
MEcDP synthase. See 2-C-Methyl-D -erythritol D -xylulose 5-phosphate (MEP/DOXP)
2,4-cyclodiphosphate (MEcDP) pathway, 50, 96, 100, 157, 158, 264,
synthase 318, 362, 458, 512
Mechanical damage, 31, 288, 290, 291, 424 Methyl jasmonate (MeJA), 22, 79, 80, 290
Mechanical injury, 166, 172, 288–290 Methyl salicylate (MeSA), 210, 267, 288, 289,
Mediterranean, 8, 12–13, 29, 78, 203, 204, 295, 298, 305, 342
217–218, 262, 269, 320, 341, 358, 359, 2-Methyltetrol, 222
364, 365, 367–372, 375, 438, 492 Methyl vinyl ketone (MVK), 222, 223
Melaleuca alternifolia, 104, 110 Mevalonate diphosphate carboxylase, 50, 99
Melitaea cinxia, 304 Mevalonate kinase, 99
Melolontha hippocastani, 27 Mevalonate (MVA) pathway, 50, 96, 98, 125,
Membrane fluidity, 48 158
Membrane stability, 220, 269 Mevalonic (MVA) acid, 50, 98, 157
Membrane stabilization, 3 pathway, 50, 121, 123–125, 157, 172
Mentha, 181–182 Michaelis-Menten constant, 327, 329, 458
Mentha x piperita, 101 Michaelis-Menten kinetics, 327
MEP/DOXP pathway (MEP pathway), 50, 51, Microbial-type terpenoid synthase, 73, 75
68, 96–97, 99–103, 108, 121, 123–128, Mitochondria, 70, 98, 102, 103, 106
130, 133–139, 142, 157–164, 166, 172, Mitochondrial respiration, 168, 172, 217, 238,
223, 264, 318, 323, 327–329, 375, 512, 264, 325
513 Model deviation, 373, 380, 409
Mesophyll diffusion resistance, 215 Model evaluation, 372, 408, 453, 461–464, 475
Metabolic response, 37, 155, 156, 171, 316 Modelling efficiency, 372–374, 377–379, 382
Metabolome, 77, 238 Model intercomparison, 380, 382, 460, 519
Metasequoia glyptostroboides, 260 Model inversion, 370, 463
Methacrolein (MAC), 222, 276 Model of Emissions of Gases and Aerosols
Methane, vi, 121, 210, 211, 254, 452, 453, from Nature (MEGAN), 186, 205, 206,
464–469, 490 319, 333, 339, 397, 399, 423, 455,
Methane lifetime, 453, 464, 467, 469 459–461, 463, 464, 474
Methanol, 13–14, 29, 120, 121, 154, 166–167, Model of Emissions of Gases and Aerosols
172, 186–191, 198, 200–203, 205, 210, from Nature2 (MEGAN2), 401, 406,
213–215, 218, 247, 249, 295, 316, 321, 409
337, 422, 455, 463, 464, 472, 514, 515 Model performance, 140, 368, 373, 377, 382,
column, 464 383, 475, 476, 518
emission, 166–167, 172, 186, 187, 198, Model validation, 372–373, 376, 378, 382
200–203, 205, 215, 316, 422, 455, 464, Model validation statistics, 372–373, 376, 378,
514 382
Methylbutenol, 55, 121, 130, 140, 214, 321 MODIS, 402–404, 417, 438
Index 541

Molecular controls, 120–121, 139 143, 223, 264, 270, 326–327, 361, 362,
Monofunctional synthase, 67, 73 457–459
Monoterpene Night time temperature, 214
emission, 12, 14, 164, 166, 186, 187, 189, NIR. See Near-infrared radiation (NIR)
192, 215–217, 220, 247, 259, 261, Nitrate (NO3 ) radicals, 286, 294–298, 306,
266–268, 270, 290, 318, 319, 326, 327, 307, 452, 493
330, 334, 339, 341, 343, 344, 358–383, Nitric acid, 287
397, 399, 403–405, 407–409, 422, 438, Nitric oxide (NO), 222, 245, 247, 297
459, 461, 474, 492, 493, 500, 510, 511, Nitric oxide synthase (NOS), 247
514, 516 Nitrogen availability, 262
fumigation, 510 Nitrogen content, 265, 422
storage, 359 Nitrogen oxides (NOx ), 96, 254, 255, 274, 285,
synthase, 52, 67, 71, 79, 130–131, 213, 297, 303, 371, 407, 416, 453, 456, 466,
214, 267, 323, 343, 344, 362–364, 378, 467, 472
510, 511 NO. See Nitric oxide (NO)
synthase activity, 214, 363, 364 NO2 , 287, 297, 299, 301
Morphological adaptation, 237, 244, 249 NOx . See Nitrogen oxides (NOx )
Morus alba, 425 Nocturnal herbivore feeding, 297
Mountain birch, 293–294 Non-emitter, vi, 8, 13, 225, 276
Mowing, 420–422 Non-emitting species, 5, 155, 271, 337, 340,
Mucuna, 422 515, 519, 520
Mucuna pruriens, 259, 263, 368–369 Non-homogeneous vegetation, 382
Multifunctional compounds, 224, 225 Non-photorespiratory respiration rate in light,
Multifunctional enzyme, 71, 225 325
Multiple selection pressures, 26 Non-specific storage, 194, 201, 214, 358, 366,
Multitrophic interactions, 22, 38, 274, 286, 375, 377, 383, 514
296, 302–303, 307, 520 Non-steady state condition, 192, 196, 197, 514
Multitrophic signalling, 285–308 Non-volatile phytohormones, 340
Mutualism, 3, 4 Norway spruce, 289, 371, 381
Mutualistic interactions, 34 Nucleation, 453, 493, 497, 498
Mycobacterium tuberculosis, 65 Numerical model, 327
Mycorrhizal association, 36 Nutrient availability, 28, 210, 258, 422
Myrcene, 25, 56–59, 71, 75, 123, 130, 131, Nutrient supply, 259
191, 295, 510
Myrcene synthase, 56, 70–72, 78, 510
O
O2 . See Oxygen (O2 )
N O3 . See Ozone
NADPH. See Nicotinamide adenine Oak, vi, 10, 11, 27, 121, 400, 405, 492
dinucleotide phosphate (NADPH) “-Ocimene, 7, 187, 188, 190, 191, 197, 203,
Nandina domestica, 425 204, 297, 298
Nash-Sutcliffe modelling efficiency, 372 Octadecanoid pathway, 31, 32
Natural aerosols, 493 Octanol/water partition coefficient, 166,
Natural CO2 spring, 258–260, 265 191–194, 203, 367
Natural selection, vi, 8, 38, 156 OH radicals, 274, 294, 295, 297, 298, 306,
Near-infrared radiation (NIR), 395, 396, 307, 383, 453, 464, 466–467, 470
400 OH-recycling, 453, 647
Nerolidol, 70, 71, 102, 106, 129 Oil glands, 22
Net ecosystem exchange (NEE), 491, 495 Oil palm. See Elaeis guineensis
Network rigidity, 160 Oilseed rape, 302
Nicotiana tabacum, 36, 61, 69, 95, 99, 106, Olea europaea, 110, 268, 269
158, 267, 268, 271 Oleoresin, 48, 78, 164–166, 318, 338
Nicotinamide adenine dinucleotide phosphate Olfactory appendages, 22
(NADPH), 51, 123, 128, 133–135, 137, Olfactory response, 299
542 Index

Olfactory sensors, 37, 297 Parasite, viii, 32, 48, 292

Ontogeny, 3, 25, 27, 28, 210, 262–263, 316, Parasitic plant, 291
317, 319 Parasitism rate, 299
Oomyzus gallerucae, 34 Parasitoid, 3, 23, 25, 27, 32–39, 287, 288,
Open-top chamber, 259, 260, 262, 268–270 293, 294, 296, 297, 299–302, 304, 305,
Opportunistic hypothesis, 11 307
Optimum temperature, 68, 70, 133, 134, 213, Particle formation, 470, 497
318, 477, 492 Particle growth, 469, 470, 497
Origanum majorana, 218 Patchoulol synthase, 99
Ornamental, 6 Pathogens, 2, 3, 9, 22, 26, 27, 48, 72, 78, 79,
Outbreaks, 35, 285, 287, 299, 402 105, 288, 290–292, 306, 320, 340
Overcrowding, 421 Pectin, 167, 172, 210, 247, 249
Overexpress, 49, 108 Pectin methylesterase (PME), 166–167, 247
Overexpression, 61, 99, 101, 106–109 Penicillium roqueforti, 65
Overflow valve hypothesis, 11 Pentalenene synthase, 65, 67, 68
Overparameterization, 377 Penumbra, 394
Oviposition, 23, 27, 30–34, 36, 37, 79, 293, PEP. See Phosphoenol pyruvate (PEP)
294 PEPc. See Phosphoenol pyruvate carboxylase
Oxidation products, 78, 239, 307, 463, 470, (PEPc)
476, 493, 498, 520 Peppermint, 101, 108
Oxidative capacity, 211, 255, 452, 503 PEP transport, 137, 265
Oxidative signalling, 341 Peroxyacetyl nitrate (PAN), 303, 453
Oxidative stress, vi, vii, 3, 12, 102, 222, 225, Phalaris arundinacea, 171
246, 270, 275, 307, 510 Phaseolus
4-Oxopinonic acid, 296–297 P. lunatus, 267, 268, 299
10-Oxopinonic acid, 296–297 P. vulgaris, 186, 187, 197
Oxygen (O2 ) “-Phellandrene, 55–57, 191, 289, 295
availability, 237, 238, 242, 516 Phellandrene synthase, 56
deficiency, 244 Phenology, 333, 420, 427
dependence, 137 2-Phenylethyl acetate, 34
deprivation, 237–238 Phenylpropanoids, 22
Oxygenated volatile, 217, 422, 515 Pheromone, 25, 33, 34, 293, 304
Oxylipins, 210 Phoenix dactylifera, 53
Ozone pH optimum, 68, 70
damage, 184 Phosphoenol pyruvate (PEP), 101, 120, 137,
flux, 271, 303 138, 160, 221, 223, 264, 265, 331, 513
fumigation, 257, 269–270, 273–274 Phosphoenol pyruvate carboxylase (PEPc),
reactions, 269 112, 113, 221, 223, 264, 265, 331, 513
removal, 472 Phosphoglyceric acid (PGA), 136, 137
tolerance, 270–271, 274 Phosphorus, 422
uptake, 184, 271, 275, 276 Photo-oxidation, vi, 469
Ozone-BVOC feedback, 472 Photorespiration, 137, 223, 325, 362
Ozone-sensitive, 267, 268 Photosynthesis, v, 2, 11–14, 51, 100, 104, 120,
123, 128, 130, 133–138, 140, 143, 157,
163, 164, 183, 185, 215, 216, 218–220,
P 222, 223, 225, 256, 266, 271, 273, 302,
Palustradiene, 60 318, 324, 327–329, 339, 362, 363, 367,
Panicum virgatum, 13 370, 377, 378, 394, 395, 434, 454, 457,
Papaya, 96 458, 470, 471, 473, 490, 491, 493–496,
Papaya ringspot virus (PRVS), 96 510–514
Parameterization, viii, 12, 204, 319, 327, 359, Photosynthetically active radiation, 455
367–368, 370, 375, 377–382, 397, 398, Photosynthetic electron transport, 51, 123, 133,
404, 407, 424–425, 427, 428, 454, 460, 223, 324, 325, 327, 328, 359, 361–364,
461, 496, 518, 519 370, 458, 513
Index 543

Photosynthetic photon flux density (PPFD), Plagiodera versicolora, 32–33

155, 167, 182, 186–188, 200–203, 324, Plant
330, 367, 393, 394, 400–401, 470–471, age, 30, 421
513 nutrition, 262
Photosynthetic rate, 183, 220, 224, 263, 471, production, 305–306
492, 513 Planted forest area, 417
Phragmites australis, 13, 259, 265, 267, 268, Plant functional type (PFT), 12, 402–404, 455,
271 461, 474
Phylloquinone, 100 Plant-herbivore interaction, 21–39, 301
Phylogenetic constraints, 24 Plant-insect interactions, 30, 31, 39, 515
Phylogenetic distance, 1 Plant-insect signalling, 29
Phylogenetic tree, 75, 104, 129, 131 Plant-to-insect-communication, 511
Phylogeny, 9 Plant-to-plant communication, 167, 515
Physcomitrella patens, 73, 75, 126 Plant-type terpenoid synthase, 66, 75
Physico chemical characteristics, 24, 29, 48, Plastid-targeting sequence, 69, 70, 128
181, 190, 194, 195, 205, 213, 366, 514, Platyprepia virginalis, 293
515 Plum, 34, 110
Physico chemical controls, vii Plutella xylostella, 300, 302
Physico chemical responses, 24 Pollinator, 3, 4, 9, 26, 30, 297
Phytohormones, 31, 340 Pollutants, 2, 13, 14, 225, 257, 258, 271, 273,
Phytophagous insects, 25 274, 276, 285–288, 294, 295, 297–304,
Phytotoxicity, 274, 306, 307 306–308, 337, 424, 427, 473, 503
Picea Ponderosa pine, 214
P. abies, 56–60, 70, 242, 289, 301, 371, Poplar. See Populus spp.
380, 381, 425, 426 Population cycles, 37
P. engelmannii, 54 Populus
P. glauca, 54, 80 P. alba, 55, 61, 162, 259, 261, 268, 269,
P. mariana, 425, 426 271
P. sitchensis, 54, 55, 57, 79, 425, 426 P. deltoides, 25, 259, 261, 262, 425
Picea spp., 110, 492 P. grandidentata, 95
’-Pinene, 28, 34, 55–58, 185, 189–191, 196, P. nigra, 25, 34, 271, 425
197, 267, 269, 295, 307, 326, 367, 405, P. tremula, 135, 214, 239, 242, 248, 425,
472 426
Pinene synthase, 56 P. tremuloides, 105, 185, 259, 261, 265,
Pinonaldehyde, 307 268, 270, 323, 368–369
Pinophyta, 5, 9, 10 P. trichocarpa, 53, 54, 63, 77, 102, 104,
Pinus 142, 425
P. contorta, 79 P. x canescens, 55, 65, 142, 169, 218, 242
P. halepensis, 217, 268, 269, 422, 426 P. x euramericana, 259, 297
P. koraiensis, 423 Populus spp., 13, 30, 49, 96, 171, 255, 419,
P. pinaster, 28, 422 440
P. pinea, 166, 187, 188, 203, 204, 341, 423 Portable photosynthesis systems, 434
P. ponderosa, 79, 214, 259, 266, 322 Post-illumination burst, 133, 135, 162
P. sabiniana, 55, 130 Post-illumination isoprene emission, 134,
P. sylvestris,31, 34, 166, 197, 242, 260, 512–513
267, 268, 270, 289, 297–298, 301, 371, Post-translational regulation, 141
380, 381, 422, 425, 426, 459, 494, 498, Predator, 3, 25, 27, 32–34, 36, 37, 48, 287,
499 288, 293, 297, 299, 304, 305, 307
P. taeda, 54, 56–58, 70, 201, 202 Predatory mite, 299
Pinus spp., 171, 492 Prenyltransferase, 51, 66, 69, 79
Pi /PEP transporter (PPT), 136 Primary aerosol, 493, 496–497, 504
Pissodes strobi, 79 Primary metabolites, 8–9, 48
Pistacia lentiscus, 217 Primary particles, 496, 502
Pixel size, 429–431 Priming, 33, 48, 290, 296, 302, 320, 511, 515
544 Index

Pristiphora abietina, 289 R

Process-based model, 454, 457, 458, 461, 473, Radar, 429, 432
474 Radiative forcing, 224, 254, 500
Procontarinia, 27–28 Radical scavenger, 298
Product specificity, 52, 71–72, 80, 131 Radiometric resolution, 429
Programmed cell death, 170, 221 Range-normalized, 373
Promoter-enhancing sequence, 80 Rare tRNA, 54
Prosopis velutina, 170 Reactive electrophilic species (RES), 221, 222,
Proteinase inhibitor protein, 80 225, 246
Protein synthesis rate, 343 Reactive nitrogen, 453
Proton-transfer reaction mass spectrometer Reactive nitrogen species (RNS), 211, 221
(PTR MS), 164, 189, 297, 434, 435, Reactive oxygen species (ROS), 3, 79, 211,
499 221–225, 247, 269, 276, 302, 515–517
Pruning, 420–422 Reactivity, 96, 120, 254, 258, 271, 276, 294,
Prunus 297, 298, 300, 383, 418, 475, 476, 517
P. avium, 110 Recombinant, 52–54, 61, 68, 158
P. domestica, 110 Redox potential, 138
Pseudotsuga menziesii, 260, 266 Regional gradient, 257
Psyllaephagus baccharidis, 293 Regional weather model, 399
Pteridophyta, 9, 10 Relative adaptiveness, 63
PTR MS. See Proton-transfer reaction mass Relaxed eddy accumulation (REA), 359,
spectrometer (PTR MS) 368–370, 374–376, 378–380, 382, 397,
Pueraria 407, 435, 462, 518
P. lobata, 61, 142, 160, 423 Remote sensing, 320, 402, 429–432, 437–438,
P. montana, 104 441, 464
Pyrus bretschneideri, 53 Repellent, 25, 26, 29, 79, 292, 293, 295, 306
Pyruvate Repress, 108, 109, 510
overflow hypothesis, 170 Repression, 109–110
transporter, 137 Residence time, 459
Pyruvate decarboxylase (PDC), 168, 170 Resin duct, 2, 79, 110, 123, 164–166, 181–182,
190, 321
Rhododendron, 292–293, 306, 420
Q Rhododendron tomentosum, 291, 306
Quantum yield, 324, 325, 335 Ribulose-1,5-bisphosphate (RuBP), 325
Quercus RNA interference (RNAi), 109, 161, 162
Q. afares, 10 RNA sequencing, 52–53
Q. alba, 320, 332 Robinia pseudoacacia, 426
Q. aliena, 425 Root compounds, 27
Q. coccifera, 367, 370, 422, 426, 492 Root mean squared error, 372–374, 378
Q. dentata, 425 ROS. See Reactive oxygen species (ROS)
Q. ilex, 8, 56, 70, 185, 186, 189, 192, 197, Rosetta host strain, 61
204, 239, 259, 260, 266–269, 271, 290, Rosmarinus officinalis, 422
297, 319, 320, 323, 326, 330, 334, 362, Rough handling, 338, 371
364, 365, 367–379, 459, 492 Rubisco, 137, 183, 325, 328, 329
Q. mongolica, 425
Q. pubescens, 259, 265, 268, 269
Q. robur, 239–243, 259, 262, 323, 422, 425 S
Q. rubra, 223, 239, 241, 242, 244, 246–247, Sabinene, 57, 191, 267
259 Saccharomyces cerevisiae, 99
Q. serrata, 425 Safety valve, 223
Q. suber, 8, 189, 369, 492 Salicylic acid (SA), 31
Q. virginiana, 184 Salinity stress, vii, 218, 219, 226
Index 545

Salix Shaded leaf area, 395, 398

S. discolor, 54, 63, 104 Shading, 6, 421, 437
S. phylicifolia, 422, 426 Shikimate pathway, 101, 210
S. sitchensis, 33 Short-term control, 155, 166, 167, 201, 205
S. viminalis, 425 Short-term fumigation, 257
Salix spp., 13, 49, 96, 255, 419, 440 Shortwave radiation, 399
Salvia officinalis, 64, 218 Signal cascades, 30, 31
Santalene/bergamotene synthase, 70 Signal cross-talk, 36
Santalene synthase, 70, 71 Signalling, vi, 22, 24, 28–33, 35–37, 39, 79,
Santalum, 71 156, 166, 167, 211, 214, 222, 224, 237,
Satellite data, 403, 404, 417, 429–432, 438, 267, 273, 285–308, 316, 340–342, 515,
462–464, 475, 476 517, 520
Satellite observations, 402, 463 Signal perception, 341
Scaling, 321, 323, 326, 329, 334, 339, 342, Silencing, 343
357–383, 393, 401, 415–441, 459, 518 Simpson’s diversity index, 418
Scattering, 224, 396, 399, 471, 490, 493, 494 Sink-source relations, 301
Scentscape, 288 SMEAR, 493–494, 497, 498
Schinus areira, 301 Snapdragon, 71, 159
Scolytid prey, 34 Soil
Scots pine, 34, 267, 289, 297, 301, 371, 381, herbivory, 36
422, 494, 498 moisture, 257, 392, 398–399, 433, 470
Screening study, 4–8 redox status, 244
Sealing of wounds, 3 water content, 339
Seasonal changes, 29, 140, 257, 331–334, 359, Soil-dwelling herbivore, 27
377–378, 456, 462 Solanum
Seasonality, 28, 140, 268, 316, 317, 331–336, S. habrochaites, 70
359, 360, 363, 364, 368, 370, 379, 423, S. lycopersicum, 159–160, 268, 269, 291
432, 455, 459 S. tuberosum, 80
Seasonal variability, 377–378 Sorghum bicolor, 200
Seasonal variation, 316, 332, 364, 365, 368, Sorghum bicolor subsp. sudanense, 200–201
371, 402, 423, 460, 469 Soybean, 142
Secondary metabolites, 2, 8, 9, 48, 98, 287, Spatial distribution, 380, 382, 474
292, 301 Spatial heterogeneity, 24
Secondary organic aerosol (SOA), vi, 49, 171, Spatial resolution, 380, 399, 400, 417, 429
211, 224, 296, 301, 358, 407, 453, Spatial scale, 27, 35, 256, 473, 517
464, 465, 469–471, 490, 491, 493, 494, Specialist(s)
496–497, 500–503, 519 feeder, 25
Secondary oxidants, 258 parasitoid, 302
Selaginella muellendorffii, 66, 73, 75–76 Species composition, 28, 406, 416, 418, 423,
•-Selinene synthase, 64 438, 492
Semivolatile compound, 48, 291, 292, 296, Spectral mixture analysis, 431
306, 469 Spectral resolution, 429, 431, 441
Senescence, 170, 333, 336, 471 SPOT, 402
Sensible heat, 171, 395, 396 Squalene-hopene synthase, 64, 67, 75
Sequence homology, 53, 68, 72, 511 Standard conditions, 136, 321, 335, 361, 365,
Serial analysis of gene expression (SAGE), 53 423, 437, 459, 475
Sesquiterpenes (SQT), 2, 3, 13, 33, 48, 51, Starch breakdown, 216
69–71, 73, 75, 78, 79, 97–100, 106, Steady-state, 126, 185, 189, 190, 196, 197,
107, 123–125, 130–132, 157–159, 210, 199, 201, 203–205, 337, 360, 366, 371,
214, 218, 224, 267, 268, 288–296, 298, 514
304, 319, 339, 421, 422, 491, 492, 511 Stereoisomer, 71
Sesquiterpene synthase, 10, 52, 54, 64, 69–71, Stereo-photography, 431
102, 105, 107, 123, 130–132, 157, 159 Stereospecific, 71, 131
Sesquiterpenoid, 50, 306 Sterol, 50, 51, 98, 99, 103
546 Index

Stomata, 132, 167, 182–184, 186, 187, 189, Taxadiene synthase, 60, 67–70, 107
190, 192, 194, 198, 200, 201, 205, 213, Taxodium distichum, 260, 266
217, 218, 243–244, 271, 459, 465, 472, Taxonomic distribution, 5
514 Taxus brevifolia, 60, 67–69
Stomatal Tectona, 440
closure, 184–186, 190, 198–201, 213, 218, Temperate, 5–6, 11, 96, 241, 320, 335, 341,
303, 383 343, 344, 402–403, 410, 492
conductance, 132, 181–206, 213, 215, 218, Temperature
240, 243, 261, 262, 271, 272, 337, 338, light dependence/dependency, 61, 132, 133,
358, 377, 394, 465, 514 155, 162, 217, 316, 318, 321, 361–363,
control, 172, 182–193, 197–201, 204, 205, 367, 368, 370, 379, 455, 459
243, 316, 337–338, 514 optimum, 68, 70, 133, 134, 213, 316, 477,
limitation, 184, 196, 205, 514–515 492
opening, 186, 189, 200, 218, 243, 492 response curve, 213, 316, 368
regulation, 185, 190, 193, 197, 472 Temporal coverage, 430, 432
resistance, 166, 167, 182, 183 Temporal resolution, 429, 435, 518
Storage emission, 266, 316, 321, 322, 371 Temporal scale, 256, 343, 399, 410
Storage pool, 3, 188–190, 192–195, 197, 201, Temporary storage pool, 188–190, 192, 193
203, 204, 213, 266, 318, 322, 337, 338, Terpene emission, 28, 97, 99, 102, 105–107,
459, 491 110, 164–166, 359, 511
Storage pool size, 338 Terpene synthase genes, 53
Streptomyces, 65, 67 Terpenoid cyclase, 66
Stress Terpenoid cyclase C-terminal domain, 66, 76
priming, 48, 342 Terpenoid cyclase N-terminal domain, 66
signalling, vi, 214, 267, 273, 342, 515, 517 Terpenoid synthase, 52–81, 109–110
threshold, 342, 518 Terpenoid synthase superfamily, 66, 76
tolerance, vi, 48, 108, 212, 219–227, 510, ’-Terpinene, 55, 57, 58, 191
511 ”-Terpinene synthase, 58
Structural alignment, 66–68 Terpinen-4-ol, 110
Structural classification of proteins (SCOP), 66 ’-Terpineol, 75, 110, 189, 191
Substrate affinity, 70 ’-Terpineol synthase, 58, 70
Substrate availability, 97, 106, 109, 134, 162, Terpinolene synthase, 58
164, 167, 171, 217, 329 Terrestrial carbon sink, 273
Substrate limitation, 155, 156, 160–164, 215, Terrestrial CLAW hypothesis, 490–493
322 Tertiary structure, 65, 68, 80
Successional gradient, 6 Tetraterpenoid, 50, 51
Sucrose synthesis, 137 Thanasimus dubius, 34
Sulphur dioxide (SO2 ), 258, 285, 287, Thelaira americana, 293
299–301, 303, 494 Thermotolerance, vi, 11, 107, 220, 225, 226,
Sulphuric acid, 287, 495, 497 272
Sunflecks, 109, 263, 395 Three-domain sesquiterpene synthase, 69, 70
Sunlit leaves, 26, 392, 393, 395 ’-Thujene, 55, 58
Surface temperature, 395, 464 Time-of-flight mass spectrometer, 142, 435
Surface to volume ratio, 198 Tocopherol, 100, 221
Susceptibility, 25, 35, 37, 102, 109, 190, 293, Top-down approach, 427, 462, 463
503 Total ecosystem respiration (TER), 491, 495
Systemic response, 340 Total leaf area, 262, 263
Systemic signal, 35 Traffic emissions, 439
Traffic flow, 436
Transcriptional control, 341, 342
T Transcriptional regulation, 97
Talking trees, 291 Transcription factor, 79
TATA box, 142 Transcription regulator, 340, 341
Taxadiene, 60, 61 Transcriptome, 49, 52–54, 77, 80, 238
Index 547

Transcriptome mining, 54, 77 V

Transcriptomics, 52–53 Vaccinium corymbosum, 290
Transfer conductance, 335, 396 van’t Hoff equation, 366
Transgenic construct, 61 Várzea, 246
Transgenic plant, 53, 219, 222, 226–227, 510, Vegetation
511 distribution, 273, 460, 461, 474, 519
Transient storage pool, 190, 203 fragmentation, 296
Transpiration rate, 183, 243, 337 Vertical variation, 379, 395, 428
Traumatic resin, 79, 166 Virtual disjunct eddy covariance, 435, 462, 518
Tree cotton. See Gossypium arboretum Vitis vinifera, 77, 110
Tree crown, 430 Volatility, vii, 8, 38, 121, 128, 166, 181–206,
Tree diversity, 418 210, 238, 360, 366, 453, 492, 494, 517
Tree inventory, 404, 433
Trichodiene synthase, 65
4,8,12-Trimethyltrideca-1,3,7,11-tetraene W
(TMTT), 288, 295, 339 Walnut, 340
Triterpenoid, 50, 65, 67 Water availability, 11, 12, 271, 337, 399, 423,
Tritrophic interactions, 22, 24, 33, 34, 36, 39, 456, 465, 492–493
299 Water solubility, 192–193, 198–199, 201,
Tritrophic patterns, 27 203–205, 295, 470
Tritrophic signalling, 288 Weather data, 398
Trophic interactions, 303 Weather effect, 140
Trophic level, 3, 30, 31, 33, 37, 286–294, 297, West Midlands, 419, 426, 427, 438, 440
299, 300, 302, 303, 307 White fir, 79
Tropical, 4–6, 96, 170, 171, 255, 266, 377, White spruce, 54, 80
397, 402, 408–410, 468, 499 Wilting point, 337
Trypsin proteinase inhibitor, 29 Within-canopy light gradient, 257
Tuberculosinol di phosphate synthase, 65 Within-canopy variation, 257, 333, 369, 370,
Turbulent diffusion, 396–397 392–393, 429, 476
Turnover rate, 218, 366, 491 Wounding, 3, 30, 31, 35, 48, 79, 80, 168–170,
Turnover time, 196–198 210, 338, 340, 422, 477
Two-big-leaf, 394, 428, 438
Type I synthase terpene, 63
Type II synthase terpene, 63
X
Xanthogaleruca luteola, 33, 34
U
Ulmus minor, 31, 34
Uncertainty analysis, 440 Y
Upscaling, 367–368, 371, 391–410 Y-tube, 299, 300
Urban area, 416–420, 429–431, 437, 439
Urban environment, 274, 416, 418, 419, 424,
432, 433, 437 Z
Urban habitat, 415–441 Zea mays, 13, 302
Urban land, 416, 432–433, 440 Zele deceptor, 294
UV radiation, 338 Ziziphus, 440